Biochem Biophys Res Commun, 1996 Jun 25, 223(3), 670 - 4 Long time-scale probing of the protein globular core using hydrogen-exchange and room temperature phosphorescence; Schlyer BD et al.; Preliminary room temperature phosphorescence measurements of the highly buried Trp109 in E . coli alkaline phosphatase have been used to report on the kinetics of protein hydrogen-deuterium exchange . Upon dilution in D2O the phosphorescence lifetime increases (at 20 degrees C) in a biphasic manner with an immediate change (< 30 seconds) followed by a slow change occurring on an extremely long timescale (days) . The immediate D2O-induced lifetime increase is similar to that observed upon dilution into glycerol, a known protein hydrating agent . On the other hand, the slow D2O-induced first order growth in Trp109 lifetime is due to exchange at highly protected protein groups . As the phosphorescence lifetime of Trp109 is dependent on local rigidity, this increase in lifetime reflects changes in alkaline phosphatase structure . This first use of room temperature phosphorescence to monitor proton exchange shows promise as a sensitive and selective probe of protein core dynamics. Biochemistry, 1996 Jun 25, 35(25), 8381 - 91 Inactivation of ribonucleotide reductase by (E)-2'-fluoromethylene-2'-deoxycytidine 5'-diphosphate: a paradigm for nucleotide mechanism-based inhibitors; van der Donk WA et al.; Ribonucleotide reductase (RDPR) from Escherichia coli catalyzes the conversion of nucleotides to deoxynucleotides and is composed of two homodimeric subunits: R1 and R2 . (E)- and (Z)-2'-fluoromethylene-2'-deoxycytidine 5'-diphosphate (FMCDP) are time dependent inactivators of this protein, with approximately 1.5 equiv being sufficient for complete loss of catalytic activity . Inactivation results from loss of the essential tyrosyl radical on R2 and alkylation of R1 . Studies using electron spin resonance spectroscopy reveal that tyrosyl radical loss is accompanied by formation of a new, substrate-based radical . Experiments using {6'-14C}-(E)-FMCDP and {5-3H}-(E)-FMCDP reveal that alkylation of R1 is accompanied by release of 0.5 equiv of cytosine and 1.4 equiv of fluoride ion . When R1 is denatured subsequent to inactivation, approximately 1 equiv of label per R1 is observed only in studies carried out with {14C}FMCDP . Under these same conditions with {3H}FMCDP, 1.5 equiv of radiolabel is detected as cytosine . Inactivation of R1 thus results from alkylation by the sugar moiety of FMCDP . While studies to isolate the alkylated amino acid on R1 were unsuccessful, studies using a variety of site-directed mutants of R1 (C462S, C225S, C754/759S, C439S, and E441Q) indicate that E441 or possibly C439 is the modified residue . Inactivation is accompanied by rapid formation of a new chromophore with a lambda max at 334 nm . Dithiothreitol does not protect the enzyme against inactivation by FMCDP, although it does prevent chromophore formation . Two possible mechanisms are proposed to accommodate these experimental observations. Biochemistry, 1996 Jun 25, 35(25), 8342 - 53 On the reactivity and ionization of the active site cysteine residues of Escherichia coli thioredoxin; Takahashi N et al.; Within various proteins of the thioredoxin family, the stability of the disulfide bond formed reversibly between the two active site cysteine residues, one accessible and one buried, varies widely and is directly correlated with the pKa value of the accessible cysteine thiol group . If applicable to thioredoxin, its stable disulfide bond would imply that its accessible thiol group should have a high pKa value, whereas it has long been considered to be about 6.7, largely on the basis of the pH dependence of its reactivity . Such kinetic data are shown to be inconsistent with known pKa values in this case; the rate constants may reflect effects in the transition state for the reaction, which is catalyzed by thioredoxin, rather than the protein itself . Ionization of the thioredoxin thiol groups was measured indirectly by the pH dependence of the equilibrium constant for their reaction with glutathione and directly by detection of the thiolate anion by its UV absorbance . Both observations indicated that both cysteine thiol groups of thioredoxin ionize with apparent pKa values in the region of 9-10 and that their ionization is not linked strongly to that of any other groups . This conclusion is not incompatible with the other data available and would make thioredoxin consistent with the relationship between thiol group ionization and disulfide stability observed in other members of the thioredoxin family. Biochemistry, 1996 Jun 25, 35(25), 8260 - 7 The role of zinc and the reactivity of cysteines in Escherichia coli primase; Griep MA et al.; Primase is the zinc metalloenzyme responsible for synthesizing RNA primers for use during DNA synthesis . To establish whether the zinc played a catalytic or structural role, the zinc was removed and the activity of the apoprimase determined . The zinc was removed with p-(hydroxymercuri)-benzenesulfonate (PMPS), which covalently reacts with cysteine sulfhydryls, EDTA was added to chelate the zinc, DTT was added to remove the PMPS from the apoprimase, and then the apoprimase was separated from the small molecules . The resulting apoprimase was fully active, indicating that the zinc played a structural role but not one involved in thermodynamic folding/unfolding . PMPS and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) cysteine reactivities indicated that the cysteines fell into three categories: one or two were fast-reacting, three were zinc-ligating, and two or three were slow or nonreacting . The major distinction between apoprimase and natural primase was that apoprimase became inactivated during storage at 4 degrees C for 10 days . Storage-induced inactivation correlated with disulfide bond formation and could be reversed by incubation with a mild reducing agent . Apoprimase oxidation also prevented zinc reconstitution which was only achieved with freshly-reduced enzyme, indicating that the zinc-ligating cysteines participated in the inactivating disulfide bonds . The conclusion was that, in natural primase, the zinc prevented disulfide bond formation which, in turn, prevented inactivation . The zinc reconstitution studies identified a strong and a weak zinc binding site . Zinc could be prevented from binding to the weak site by the presence of magnesium, indicating that the weak site may be the catalytic magnesium site in which two of the seven cysteines were located. Biochemistry, 1996 Jun 25, 35(25), 8252 - 9 The synthetic/editing active site of an aminoacyl-tRNA synthetase: evidence for binding of thiols in the editing subsite; Jakubowski H; The active site of methionyl-tRNA synthetase (MetRS) possesses two functions: synthetic, which provides Met-tRNA for protein synthesis, and editing, which rejects inadvertently misactivated homocysteine . During editing, the side chain -SH group of homocysteine reacts with its activated carboxyl group forming a cyclic thioester, homocysteine thiolactone . As shown here, the side chain -SH and the activated carboxyl groups do not need to be present on the same molecule for the editing to occur . Thioester formation occurs when a thiol and activated methionine, in the form of Met-tRNA, are incubated with MetRS . Depending on the structure of thiols, methionine thioesters may undergo secondary acyl transfer reactions to cis amino, hydroxy, or carboxyl groups which yield methionine dipeptides, esters, or anhydrides, respectively . At saturating thiol concentrations, formation of some thiol derivatives of methionine is as fast as formation of homocysteine thiolactone . Thiol specificity of the reaction and noncompetitive inhibition by the cognate methionine, as well as structure-function studies of active site MetRS mutants, all indicate that there is a specific -SH binding subsite, distinct from the methionine binding subsite, in the synthetic/editing active site of MetRS. Biochemistry, 1996 Jun 25, 35(25), 8181 - 6 Effects of mutagenesis of aspartic acid residues in the putative phosphoribosyl diphosphate binding site of Escherichia coli phosphoribosyl diphosphate synthetase on metal ion specificity and ribose 5-phosphate binding; Willemoes M et al.; The three conserved aspartic acid residues of the 5-phospho-D-ribosyl alpha-1-diphosphate binding site (213-GRDCVLVDDMIDTGGT-228) of Escherichia coli phosphoribosyl diphosphate synthetase were studied by analysis of the mutant enzymes D220E, D220F, D221A, D224A, and D224S . The mutant enzymes showed an increase in KM for ribose 5-phosphate in the presence of at least one of the divalent metal ions Mg2+, Mn2+, Co2+, or Cd2+, with the most dramatic changes revealed by the D220E and D220F enzymes in the presence of Co2+ and the D221A enzyme in the presence of Mn2+ or Co2+ . The D220F and D221A enzymes both showed large decreases in Vapp in the presence of the various divalent metal ions, except for the D221A enzyme in the presence of Co2+ . Vapp of the D220E enzyme was similar to that of the wild-type enzyme in the presence of Mg2+, Mn2+, or Cd2+, whereas the Vapp was increased in the presence of Co2+ . Vapp values of the D224A and D224S enzymes were lowered to 10-15-fold and 3-4-fold in the presence of Mg2+ or Mn2+, respectively, whereas Vapp was similar to that of the wild-type and KM for Rib-5-P was increased 4-fold in the presence of Cd2+ . The changes in KM for ribose 5-phosphate and Vapp of the mutant enzymes were dependent on the metal ion present, suggesting a function of the investigated aspartic acid residues both in the binding of ribose 5-phosphate, possibly via a divalent metal ion, and in the interaction with a divalent metal ion during catalysis. Mol Gen Genet, 1996 Jun 24, 251(4), 499 - 502 Molecular cloning and sequence analysis of a cDNA encoding a cysteine proteinase inhibitor from Sorghum bicolor seedlings; Li Z et al.; A 711-bp cDNA encoding a cysteine proteinase inhibitor (cystatin) was isolated from a cDNA library prepared from 7-10 cm Sorghum bicolor seedlings . The nearly full-length cDNA clone encodes 130 amino acid residues, which include the Gin-Val-Val-Ala-Gly motif, conserved among most of the known cystatins as a probable binding site for cysteine proteinases . The amino acid sequence of sorghum cystatin deduced from the cDNA clone shows significantly homology to those of other plant cystatins . The sorghum cystatin expressed in E . coli showed a strong papain-inhibitory activity. Mol Gen Genet, 1996 Jun 24, 251(4), 493 - 8 Analysis of the mutagenic properties of the UmuDC, MucAB and RumAB proteins, using a site-specific abasic lesion; Lawrence CW et al.; The mucAB and rumAB loci have been shown to promote mutagenesis to a greater extent than the structurally and functionally homologous Escherichia coli umuDC operon . We have analyzed the basis of this enhanced mutagenesis by comparing the influence of these operons, relative to umuDC, on the mutagenic properties of each of two abasic sites, specifically located in a single-stranded vector . Experiments with these vectors are useful analytical tools because they provide independent estimates of the efficiency of translesion synthesis and of the relative frequencies of each type of nucleotide insertion or other kind of mutagenic event . The umuDC, mucAB, and rumAB genes were expressed from their natural LexA-regulated promoter on low-copy-number plasmids in isogenic strains carrying a umuDC deletion . In addition, plasmids expressing the UmuD'C, MucA'B, or RumA'B proteins were also used . Compared to umuDC, the chief effect of mucAB was to increase the efficiency of translesion synthesis past the abasic site . The enhanced capacity of mucAB for translesion synthesis depended about equally on an inherently greater capacity to promote this process and on a greater susceptibility of the MucA protein to proteolytic processing . The RumA protein also appeared to be more susceptible to proteolytic processing, but the inherent capacity of the Rum products for translesion synthesis was no greater than that of UmuDC . dAMP was inserted opposite one of the two abasic sites studied at a somewhat greater frequency in strains expressing rum (82%) compared to those expressing umu (72%), which might result in higher mutation frequencies in rumAB than in umuDC strains. Mol Gen Genet, 1996 Jun 24, 251(4), 407 - 11 Two overlapping SOS-boxes in ColE operons are responsible for the viability of cells harboring the Col plasmid; Lu FM et al.; In this study, oligonucleotide-directed site-specific mutagenesis was used to change the consensus sequences of the LexA binding motifs in either one of the two SOS-boxes of the ColE7 operon . The results indicated that both mutants produced larger amounts of colicin than cells harboring the wild-type ColE7 plasmid . This finding would imply that two biologically functional SOS boxes exist in the ColE7 operon . In the non-induced state, no lysis of cells harboring wild-type plasmids occurred at 37 degrees C, whereas, cells harboring recombinant plasmids containing either one of the mutated SOS boxes underwent lysis within 100 min under the same conditions . This result indicated that adaptation of two SOS boxes of the ColE operon would obviously tightly control the expression of ColE operons . In such a way that it may prevent excessive expression of the lysis (cel) gene, thus safeguard the host cells from being lysed in ordinary living conditions. FEBS Lett, 1996 Jun 24, 389(1), 32 - 4 The Escherichia coli trigger factor; Hesterkamp T et al.; E . coli trigger factor is an abundant cytosolic protein originally identified by its ability to maintain the precursor of a secretory protein in a translocation competent form . Recent studies shed new light on the function of this protein . Trigger factor was found to be a peptidyl-prolyl-cisltrans-isomerase capable of catalysing protein folding in vitro, to associate with nascent cytosolic and secretory polypeptide chains, and to cooperate with the GroEL chaperone in promoting proteolysis of an unstable polypeptide in vivo . These findings suggest roles for trigger factor in various folding processes of secretory as well as cytosolic proteins. J Mol Biol, 1996 Jun 21, 259(4), 792 - 804 1H, 15N resonance assignment and three-dimensional structure of CYP1 (HAP1) DNA-binding domain; Timmerman J et al.; CYP1(HAP1) is a transcriptional activator involved in the aerobic metabolism of the yeast Saccharomyces cerevisiae . The amino acid sequence of its DNA-binding domain suggests that it belongs to the "zinc cluster" class . This region is indeed characterized by a pattern known to form a bimetal thiolate cluster where two zinc ions are coordinated by six cysteine residues . Structures of two such domains, those from GAL4 and PPR1, have been solved as complexes with DNA . These domains consist of the zinc cluster connected to a dimerization helix by a linker peptide . They recognize, as a dimer, an inverted repeat of a CGG motif that is separated by a specific number of bases . Interestingly, the specificity of that interaction seems not to be due to the interaction between the cluster region and the DNA but rather to a fine tune between the structure of the linker peptide and the number of base-pairs separating the two CGGs . However, the CYP1 target sites fail to display such a consensus sequence . One of the two CGG sites is poorly conserved and some experiments suggest a direct rather than an inverted repeat . Using 1H, 15N and 113Cd NMR spectroscopy, we have undertaken the analysis of the structural properties of the CYP1(56-126) fragment that consists of the zinc-cluster region, the linker peptide and a part of the dimerization helix . We have demonstrated that the six cysteine residues of the peptide chelate two cadmium ions as in GAL4 and PPR1 . Fifteen structures of the zinc-cluster region (residues 60 to 100) were calculated, the linker peptide and the dimerization helix being unstructured under the conditions of our study . This region possesses the same overall fold as in GAL4 and PPR1, and most of the side-chains involved in the interaction with DNA are structurally conserved . This suggests that the CYP1 zinc-cluster region recognizes a CGG triplet in the same way as GAL4 and PPR1 . In this case, the particular properties of CYP1 seem to be due to the structure of the linker peptide and/or of the dimerization helix. J Mol Biol, 1996 Jun 21, 259(4), 761 - 73 Refined structure of lac repressor headpiece (1-56) determined by relaxation matrix calculations from 2D and 3D NOE data: change of tertiary structure upon binding to the lac operator; Slijper M et al.; The solution structure of the DNA binding domain of lac repressor (headpiece 1-56; HP56) has been refined using data from 2D and 3D NMR spectroscopy . The structure was derived from 1546 restraints (giving an average of 27.6 per residue), comprising 389 intraresidual, 402 sequential, 385 medium range and 325 long range distance restraints and also 30 phi and 15 chi 1 dihedral angle restraints . The structures were determined by the method of direct refinement against nuclear Overhauser enhancement peak volumes with the program DINOSAUR . The final set of 32 selected structures displayed an r.m.s . deviation from the average of 0.43(+/-0.08) A angstroms (backbone) and 0.95(+/-0.08) angstroms (all heavy atoms) for the best defined region of the protein (residues 3 to 49) . The ensemble R-factor was 0.35, which indicates close correspondence with the experimental data . The structures revealed good stereochemical qualities . The conformations of the NMR structures of free and DNA complexed lac repressor headpiece were compared . The regions comprising the secondary structure elements show close correspondence for both conformations . However, the conformation of the loop between helix II and III changes considerably upon complexation of the headpiece . This change in the conformation of the loop in lac HP56 is essential for binding of the side-chains of residues Asn25 and His29 to the lac operator DNA . Finally, the lac headpiece residues that are intolerant to mutations were analysed . Most of these mutation-sensitive residues are important for a correct folding of the headpiece region, and a number of these residues are also involved in contacting the operator DNA. J Mol Biol, 1996 Jun 21, 259(4), 575 - 8 Interaction between cAMP-dependent protein kinase catalytic subunit and peptide inhibitors analyzed with lambda repressor fusions; Jappelli R et al.; The lambda phage repressor is currently used as a genetic tool to analyze homodimeric interactions in Escherichia coli . We have applied this system to detect the interaction that takes place within an enzyme-protein inhibitor complex . The sequences encoding the catalytic subunit of the cAMP-dependent protein kinase and the active portion of the natural thermostable protein kinase inhibitor have been fused to the carboxy terminus of the repressor DNA binding domain and introduced into compatible plasmids . Co-expression of the two gene fusions in E . coli lead to the formation of heterodimers that confer a high level of protection from lambda phage infection . The level of lambda immunity depends specifically upon the amino acid sequence of the interacting proteins, as a single amino acid substitution in the inhibitor peptide (Phe10-Ala) restores the sensitivity phenotype. J Biol Chem, 1996 Jun 21, 271(25), 14665 - 7 Premature chromatin condensation induced by loss of RCC1 is inhibited by GTP- and GTPgammaS-Ran, but not GDP-Ran; Ohba T et al.; RCC1 is a guanine nucleotide exchanging factor acting on nuclear G protein Ran . Premature chromatin condensation occurs in the temperature-sensitive rcc1- mutant of the BHK21 cell line, tsBN2, at the restrictive temperature . This observation can be explained if the premature activation of MPF is normally inhibited by GTP-Ran . In the absence of RCC1, GDP-Ran predominates, resulting in MPF activation . However, experiments with Ran mutants to determine whether GTP- or GDP-Ran prevents activation of MPF have yielded conflicting results . In order to clarify this point, we have microinjected nucleotide-bound Ran, instead of mutated Ran, into the nuclei of tsBN2 cells treated to reduce RCC1-mediated guanine nucleotide exchange . GTP-Ran, GTPgammaS-Ran, and GDP-Ran all inhibited chromatin condensation . However, the inhibition of chromatin condensation by GDP-Ran could be completely abolished by co-injection with GDP, but not GTP . Thus, we conclude that GTP-Ran blocks the activation of MPF and that hydrolysis of GTP is not required to prevent MPF activation. J Biol Chem, 1996 Jun 21, 271(25), 14676 - 9 Characterization of glycosaminoglycan-binding domains present in insulin-like growth factor-binding protein-3; Fowlkes JL et al.; Matrix metalloproteinase 3 cleaves insulin-like growth factor-binding protein-3 (IGFBP-3) into six fragments, four of which bind heparin-Sepharose (Fowlkes, J . L., Enghild, J . J., Suzuki, K., and Nagase, H . (1994) J . Biol . Chem . 269, 25742-25746) . Sequence analysis of IGFBP-3 heparin-binding fragments shows that all fragments contain at least one of two highly basic, putative heparin-binding consensus sequences present in IGFBP-3 . Epitope-specific antibodies generated against synthetic peptides containing these domains recognized IGFBP-3, yet were significantly inhibited from binding in the presence of heparin, demonstrating that these regions of IGFBP-3 contain functional heparin-binding domains . IGFBP-3 peptides containing one of the two heparin-binding consensus sequences bound heparin in a solid phase binding assay in a dose-dependent and saturable manner . However, the IGFBP-3 peptide containing the heparin-binding consensus sequence 149KKGHA153 bound heparin with approximately 4-fold less affinity than the IGFBP-3 peptide containing the longer heparin-binding consensus sequence 219YKKKQCRP226 . Examination of several well characterized glycosaminoglycans to inhibit the binding of heparin to both heparin-binding IGFBP-3 peptides revealed that the most potent inhibitors were heparin, heparan sulfate, and dermatan sulfate; chondroitin sulfate A and hyaluronic acid were intermediate in their inhibitory activities; and chondroitin sulfate C caused no inhibition . These studies identify and characterize the glycosaminoglycan-binding domains in IGFBP-3, providing a basis for the better understanding of IGFBP-3-glycosaminoglycan interactions at the cellular and extracellular interface. J Biol Chem, 1996 Jun 21, 271(25), 15034 - 44 The mammalian immediate-early TIS21 protein and the leukemia-associated BTG1 protein interact with a protein-arginine N-methyltransferase; Lin WJ et al.; The TIS21 immediate-early gene and leukemia-associated BTG1 gene encode proteins with similar sequences . Two-hybrid analysis identified a protein that interacts with TIS21 and BTG1 . Sequence motifs associated with S-adenosyl-L-methionine binding suggested this protein might have methyltransferase activity . A glutathione S-transferase (GST) fusion of the putative methyltransferase modifies arginine residues, in appropriate protein substrates, to form NG-monomethyl and NG,NG-dimethylarginine (asymmetric) . We term the protein- arginine N-methyltransferase (EC 2.1.1.23) gene "PRMT1, " for protein-arginine methyltransferase 1 . GST-TIS21 and GST-BTG1 fusion proteins qualitatively and quantitatively modulate endogenous PRMT1 activity, using control and hypomethylated RAT1 cell extracts as methyl-accepting substrates . PRMT1 message appears ubiquitous, and is constitutive in mitogen-stimulated cells . Modulation of PRMT1 activity by transiently expressed regulatory subunits may be an additional mode of signal transduction following ligand stimulation. J Biol Chem, 1996 Jun 21, 271(25), 14891 - 6 Rescue of the catalytic activity of an H42A mutant of horseradish peroxidase by exogenous imidazoles; Newmyer SL et al.; His-42 plays a critical role in the H2O2-dependent catalytic turnover of horseradish peroxidase (HRP) . This is clearly illustrated by the finding that an H42A mutation decreases the rate of Compound I formation by a factor of approximately10(6) . As shown here, the addition of 2-substituted imidazoles partially rescues both the rate of formation of Compound I and the peroxidase activity of the H42A mutant . 2-Substituted imidazoles are the most effective because they do not coordinate to the iron . In contrast to native HRP, which exhibits a parabolic pH profile, and the H42A mutant, for which the activity increases linearly with increasing pH, the activity of the H42A mutant in the presence of 1,2-dimethylimidazole (pKa = 8.0) exhibits a sigmoidal pH dependence with a midpoint at pH 8.0 +/- 0.2 . Similar results are obtained with 2-methylimidazole . These results establish that the free base forms of these imidazoles facilitate HRP turnover . The spectroscopic binding constants for 1, 2-dimethylimidazole and 2-methylimidazole are Kd = 2.9 +/- 1.3 and 2 . 5 +/- 0.2 M, respectively . When cyanide is bound to the heme, the Kd for 1,2-dimethylimidazole is 0.17 M . This >10-fold decrease in Kd may reflect hydrogen bonding of the protonated imidazole to the iron-coordinated cyanide . The log of the rate of Compound I formation exhibits a linear dependence on the molecular volume of the imidazoles used to rescue the activity . If the rates are corrected for differences in the size of the imidazoles, the log of the rates is linearly related to the pKa of the imidazoles . This Bronsted analysis predicts that approximately 60% of a positive charge develops on the imidazole in the transition state of Compound I formation . The results confirm the acid-base role of the distal histidine, demonstrate that exogenous histidines can function as surrogates for the missing histidine in the H42A mutant, and provide a transition state model of relevance to the formation of Compound I in the native protein. J Biol Chem, 1996 Jun 21, 271(25), 14734 - 9 Expression and spectroscopic characterization of the hydrogenosomal {2Fe-2S} ferredoxin from the protozoan Trichomonas vaginalis; Vidakovic MS et al.; The heterologous expression and spectroscopic characterization of the {2Fe-2S} ferredoxin from the sexually transmitted human parasite Trichomonas vaginalis is described . Using oligonucleotide primers based on the deduced DNA sequence, the gene encoding the ferredoxin was amplified by polymerase chain reaction and cloned into a T7 RNA polymerase expression vector . Expression of the gene in Escherichia coli host HMS174(DE3) resulted in the high level production of the protein with the correctly assembled iron-sulfur cluster . The absorption, circular dichroism, resonance Raman, and EPR spectra of the recombinant protein revealed many differences from those of other {2Fe-2S} ferredoxins . The redox potential of the protein (-347 mV versus normal hydrogen electrode) was also determined . Whereas the amino acid sequence of T . vaginalis ferredoxin showed greatest homology to the {2Fe-2S} ferredoxins found in bacteria and vertebrate mitochondria which function in cytochrome P450 oxidation pathways, the spectroscopic properties showed substantial dissimilarity . Differences in the biophysical properties and function of T . vaginalis ferredoxin are proposed to result from the characteristic amino acid sequence of the parasite protein near the cysteine residues that ligate the valence-localized Fe(III) site of the reduced cluster. J Biol Chem, 1996 Jun 21, 271(25), 15244 - 52 Influence of specific signal peptide mutations on the expression and secretion of the alpha-amylase inhibitor tendamistat in Streptomyces lividans; Fass SH et al.; The Streptomyces alpha-amylase inhibitor tendamistat is secreted by a signal peptide with an amino-terminal charge of +3 . To elucidate the influence of the charged residues on protein secretion in Streptomyces, the amino-terminal charge was varied from +6 to neutral net charge . The effects of charge variation were analyzed in combination with three Streptomyces promoters and two transcriptional terminators . Introduction of additional positive charges significantly decreased the amount of secreted tendamistat . On the contrary, a charge reduction to +2 resulted in the doubling of inhibitor production . After exclusion of transcriptional effects, the observed alterations of inhibitor secretion by the mutants with a charge of +6 to +2 were attributed to a modulation of precursor synthesis . Furthermore, a tight coupling of synthesis and export was stated . Charge reduction to +1 or neutral charge generally reduced the yield of secreted tendamistat, yet remarkable differences were found for mutants with identical net charge . Elimination of the positive charge at a defined position resulted in the release of tendamistat precursor protein, which suggested a specific uncoupling of synthesis and translocation. J Biol Chem, 1996 Jun 21, 271(25), 14903 - 9 Cloning and functional characterization of the gene encoding the TFIIIB90 subunit of RNA polymerase III transcription factor TFIIIB; Roberts S et al.; The yeast RNA polymerase III (pol III) general transcription factor TFIIIB is composed of three subunits; the TATA-binding protein (TBP)1, the TFIIB-related factor (BRF1), and a third factor termed TFIIIB90 or B" . Here we report the purification of yeast TFIIIB90, cloning of the gene encoding TFIIIB90, and reconstitution of TFIIIB from recombinant polypeptides . The TFIIIB90 open reading frame encodes a 68-kDa polypeptide and has no obvious similarity to any other known protein sequences . The gene encoding TFIIIB90 is essential for viability of yeast . Using recombinant TFIIIB subunits, we found that TFIIIB90 interacts weakly with TBP in the absence of BRF1, and that this interaction is enhanced at least 25-fold by BRF1 . In addition, TFIIIB90 showed pol III specificity as it could not interact with the pol II-specific TFIIB-TBP-DNA complex . To localize the regions of the TBP-DNA complex that interact with BRF1 and TFIIIB90, we tested whether the pol II factors TFIIA and TFIIB interfered with the binding of BRF1 and TFIIIB90 to TBP-DNA . Our results suggest that the binding sites for BRF1 and TFIIIB90 on TBP-DNA both overlap the binding sites for TFIIA and TFIIB. J Biol Chem, 1996 Jun 21, 271(25), 14747 - 53 Specificity and kinetics of haloalkane dehalogenase; Schanstra JP et al.; Haloalkane dehalogenase converts halogenated alkanes to their corresponding alcohols . The active site is buried inside the protein and lined with hydrophobic residues . The reaction proceeds via a covalent substrate-enzyme complex . This paper describes a steady-state and pre-steady-state kinetic analysis of the conversion of a number of substrates of the dehalogenase . The kinetic mechanism for the "natural" substrate 1,2-dichloroethane and for the brominated analog and nematocide 1,2-dibromoethane are given . In general, brominated substrates had a lower Km, but a similar kcat than the chlorinated analogs . The rate of C-Br bond cleavage was higher than the rate of C-Cl bond cleavage, which is in agreement with the leaving group abilities of these halogens . The lower Km for brominated compounds therefore originates both from the higher rate of C-Br bond cleavage and from a lower Ks for bromo-compounds . However, the rate-determining step in the conversion (kcat) of 1, 2-dibromoethane and 1,2-dichloroethane was found to be release of the charged halide ion out of the active site cavity, explaining the different Km but similar kcat values for these compounds . The study provides a basis for the analysis of rate-determining steps in the hydrolysis of various environmentally important substrates. J Biol Chem, 1996 Jun 21, 271(25), 15230 - 6 Alteration of enzyme function of the type II hexokinase C-terminal half on replacements of restricted regions by corresponding regions of glucokinase; Kogure K et al.; To know the structural properties responsible for the enzymic activity of the 50-kDa C-terminal half of type II hexokinase (HKII-C) derived from rat hepatoma cell line AH130, we constructed cDNAs of HKII-C and its recombinants in which restricted regions containing highly conserved sequences, referred to as regions 2 and 3, were replaced by the corresponding regions of glucokinase . The binding domains of ATP and glucose were proposed to exist in these regions, respectively . Then, the HKII-C and chimera HKII-Cs were overexpressed in Escherichia coli BL21(DE3)pLysS . They all exhibited hexokinase activity, and their activities were inhibited by glucose-6-phosphate (Glc-6-P) competitively for ATP and uncompetitively for glucose . The replacement of region 2 of HKII-C by the corresponding region of glucokinase increased the affinity for glucose and decreased the affinity for Glc-6-P, but it did not significantly affect the affinity for ATP . In contrast, the replacement of region 3 did not cause an appreciable change in hexokinase activity . These findings suggest that region 2 is associated with the binding of ATP and Glc-6-P, and that the latter binding site is located close to the ATP binding site . In addition, region 2 was suggested to be directly related with the binding of glucose and other hexoses. J Biol Chem, 1996 Jun 21, 271(25), 14819 - 24 Purification by Ni2+ affinity chromatography, and functional reconstitution of the transporter for N-acetylglucosamine of Escherichia coli; Mukhija S et al.; The N-acetyl-D-glucosamine transporter (IIGlcNAc) of the bacterial phosphotransferase system couples vectorial translocation to phosphorylation of the transported GlcNAc . IIGlcNAc of Escherichia coli containing a carboxyl-terminal affinity tag of six histidines was purified by Ni2+ chelate affinity chromatography . 4 mg of purified protein was obtained from 10 g (wet weight) of cells . Purified IIGlcNAc was reconstituted into phospholipid vesicles by detergent dialysis and freeze/thaw sonication . IIGlcNAc was oriented randomly in the vesicles as inferred from protein phosphorylation studies . Import and subsequent phosphorylation of GlcNAc were measured with proteoliposomes preloaded with enzyme I, histidine-containing phosphocarrier protein, and phosphoenolpyruvate . Uptake and phosphorylation occurred in a 1:1 ratio . Active extrusion of GlcNAc entrapped in vesicles was also measured by the addition of enzyme I, histidine-containing phosphocarrier protein, and phosphoenolpyruvate to the outside of the vesicles . The Km for vectorial phosphorylation and non-vectorial phosphorylation were 66 . 6 +/- 8.2 microM and 750 +/- 19.6 microM, respectively . Non-vectorial phosphorylation was faster than vectorial phosphorylation with kcat 15.8 +/- 0.9 s-1 and 6.2 +/- 0.7 s-1, respectively . Using exactly the same conditions, the purified transporters for mannose (IIABMan, IICMan, IIDMan) and glucose (IICBGlc, IIAGlc) were also reconstituted for comparison . Although the vectorial transport activities of IICBAGlcNAc and IICBGlc . IIAGlc are inhibited by non-vectorial phosphorylation, no such effect was observed with the IIABMan.IICMan.IIDMan complex . This suggests that the molecular mechanisms underlying solute transport and phosphorylation are different for different transporters of the phosphotransferase system. J Biol Chem, 1996 Jun 21, 271(25), 14754 - 63 On the role of helix 0 of the tryptophan synthetase alpha chain of Escherichia coli; Yee MC et al.; The role of helix 0 of the alpha chain (TrpA) of the tryptophan synthetase alpha2beta2 multi-functional enzyme complex of Escherichia coli was examined by deleting amino-terminal residues 2-6, 2-11, or 2-19 of TrpA . Selected substitutions were also introduced at TrpA positions 2-6 . The altered genes encoding these polypeptides were overexpressed from a foreign promoter on a multicopy plasmid and following insertion at their normal chromosomal location . Each deletion polypeptide was functional in vivo . However all appeared to be somewhat more labile and insoluble and less active enzymatically than wild type TrpA . The deletion polypeptides were overproduced and solubilized from cell debris by denaturation and refolding . Several were partially purified and assayed in various reactions in the presence of tryptophan synthetase beta2 (TrpB) . The purified TrpADelta2-6 and TrpADelta2-11 deletion polypeptides had low activity in both the indole + serine --> tryptophan reaction and the indoleglycerol phosphate + serine --> tryptophan reaction . Poor activity in each reaction was partly due to reduced association of TrpA with TrpB . The addition of the TrpA ligands, alpha-glycerophosphate or indoleglycerol phosphate, during catalysis of the indole + serine --> tryptophan reaction increased association and activity . These findings suggest that removal of helix 0 of TrpA decreases TrpA-TrpB association as well as the activity of the TrpA active site . Alignment of the TrpA sequences from different species indicates that several lack part or all of helix 0 . In some of these polypeptides, extra residues at the carboxyl end may substitute for helix 0. Science, 1996 Jun 21, 272(5269), 1802 - 4 Punctuated evolution caused by selection of rare beneficial mutations; Elena SF et al.; For more than two decades there has been intense debate over the hypothesis that most morphological evolution occurs during relatively brief episodes of rapid change that punctuate much longer periods of stasis . A clear and unambiguous case of punctuated evolution is presented for cell size in a population of Escherichia coli evolving for 3000 generations in a constant environment . The punctuation is caused by natural selection as rare, beneficial mutations sweep successively through the population . This experiment shows that the most elementary processes in population genetics can give rise to punctuated evolution dynamics. Biochemistry, 1996 Jun 18, 35(24), 8009 - 17 Electrogenic K+ transport by the Kdp-ATPase of Escherichia coli; Fendler K et al.; Charge translocation by the Kdp-ATPase of Escherichia coli was measured by adsorption of proteoliposomes to a planar lipid membrane . The proteoliposomes were prepared by reconstitution of purified Kdp-ATPase into liposomes prepared from E . coli lipids . The protein was activated by a ATP concentration jump produced by photolysis of a protected derivative of ATP, caged ATP . Charge translocation was measured with a time resolution of 15-40 ms . Stationary currents demonstrated the continuous pumping activity of the enzyme . Control measurements with the potential-sensitive dye DiSC3(5) showed a negative potential inside the proteoliposomes after activation with ATP . The measured electrical signals as well as the dye measurements correspond to the transport of positive charge to the intracellular face of the protein . The electrical signal was increased when K+ was inside the proteoliposomes (K0.5 approximately 50 microM) and was inhibited by vanadate . These experiments demonstrate the electrogeneity of the Kdp-ATPase in a purified reconstituted system. Biochemistry, 1996 Jun 18, 35(24), 7968 - 73 An unnatural folate stereoisomer is catalytically competent in DNA photolyase; Lipman RS et al.; The folate chromophore in native Escherichia coli DNA photolyase ({6R}-5,10-CH+-H4Pte(Glu)n=3-6) serves as an antenna, transferring light energy to the fully reduced flavin (FADH2) reaction center at high efficiency (EET = 0.92) . Apophotolyase reconstituted after an overnight incubation with {6R,S}-5,10-CH+-H4folate (a monoglutamate analogue of the native cofactor) contains equimolar amounts of the {6R}- and {6S}-isomers, suggesting similar binding affinities . A rapid, biphasic increase in fluorescence (approximately 100-fold) is observed upon binding of 5,10-CH+-H4folate to apophotolyase at 5 degrees C; the {6S}-isomer binds about 25-fold faster than the {6R}-isomer . Although identical absorption and fluorescence emission maxima are observed for enzyme reconstituted with {6S}-, {6R}-, or {6R,S}-5,10-CH+-H4folate, folate fluorescence quantum yield values vary depending on the stereochemical configuration at the 6 position (theta = 0.18, 0.82, or 0.46, respectively, at 5 degrees C), a feature not seen with free folate . The fluorescence of enzyme-bound folate is quenched upon flavin binding; the efficiency of quenching by flavin radical (EQ = 0.96) or FADH2 (EQ = 0.89) is the same for both folate isomers . In contrast, energy transfer from folate to FADH2 is sensitive to the stereochemical configuration at the 6 position . The efficiency of energy transfer observed for enzyme containing FADH2 and {6S}-, {6R}-, or {6R,S}-5,10-CH+-H4folate (theta = 0.26, 0.66, or 0.44, respectively) is directly proportional to the fluorescence quantum yield observed for folate in the absence of FADH2, as expected for Forster-type energy transfer . Although less efficient, the unnatural {6S}-isomer is catalytically functional, a feature not previously observed with other folate-dependent enzymes . Fluorescence quantum yield studies at 77 K with free (theta = 0.67) and enzyme-bound (theta = 1.0) folate suggest that differences in solvent exposure may contribute to the fluorescence efficiency differences observed with the enzyme-bound folate isomers at 5 degrees C. Biochemistry, 1996 Jun 18, 35(24), 7895 - 902 Rabbit muscle creatine kinase: consequences of the mutagenesis of conserved histidine residues; Chen LH et al.; Creatine kinase (CK; EC 2.7.3.2) catalyzes the reversible conversion of creatine and MgATP to phosphocreatine and MgADP . In the absence of an X-ray crystal structure, we have used the sequence homology of creatine kinases and other guanidino kinases from a variety of sources to identify the conserved histidine residues in rabbit muscle CK, as well as to try to pinpoint a reactive histidine that has been implicated in the active site . This residue has been proposed to act as a general acid/base catalyst assisting in the phosphoryl transfer mechanism {Cook et al . (1981) Biochemistry 20, 1204-1210} . There are 17 histidine residues in rabbit muscle CK, and of these, only five have been conserved in all guanidino kinase sequences published to date {Muhlebach et al . (1994) Mol . Cell . Biochem . 133, 245-62} . In rabbit muscle CK, these residues are H96, H105, H190, H233, and H295 . We have carried out site-specific mutagenesis of these five histidine residues, replacing each with an asparagine . Each of these mutants exhibited enzymatic activity but to varying degrees . The H105N, H190N, and H233N mutants displayed specific activities similar to that of the wild-type enzyme . H96N has high activity, but appears to be quite unstable, losing catalytic activity upon cell lysis by sonication and/or chromatographic steps involved in purification . H295N shows a significantly reduced catalytic activity relative to the native enzyme, due to marked decreases in kcat and the affinities for both substrates . Each of the five mutants is inactivated by diethyl pyrocarbonate (DEP), and inactivation is reversible upon incubation with hydroxylamine . However, only H295N shows a dramatically reduced rate of inactivation relative to native CK, consistent with H295 being the residue modified by DEP in the native enzyme . These intriguing results indicate that four of the conserved histidines (H96, H105, H295, and H233) are not essential for activity, and while H295 may be at the active site of CK, it is unlikely to play the role of a general acid/base catalyst. Biochemistry, 1996 Jun 18, 35(24), 7873 - 8 Expression and gene disruption analysis of the isocitrate dehydrogenase family in yeast; Zhao WN et al.; Mammalian and yeast cells contain three isozymes of isocitrate dehydrogenase: mitochondrial NAD- and NADP-specific enzymes and a cytosolic NADP-specific enzyme . Independent metabolic functions of these enzymes in Saccharomyces cerevisiae were examined by analyses of expression and of phenotypes displayed by mutants containing all possible combinations of isozyme gene disruptions . All three isocitrate dehydrogenases are expressed at high levels with growth on nonfermentable carbon sources, whereas the mitochondrial NADP-specific enzyme constitutes the major cellular activity with growth on glucose . Distinct growth phenotypes are observed for mutants expressing a single isozyme, and expression of at least one isozyme is necessary for glutamate-independent growth . The NADP-specific tricarboxylic acid cycle isocitrate dehydrogenase from Escherichia coli was expressed in mitochondrial and cytosolic compartments of the yeast disruption mutants using plasmids carrying gene fusions of yeast promoters and a mitochondrial targeting presequence with the bacterial coding sequence . The bacterial enzyme is competent for restoration of NADP-specific functions in either compartment but does not compensate for function of the yeast NAD-specific tricarboxylic acid cycle enzyme. Biochemistry, 1996 Jun 18, 35(24), 7787 - 93 Escherichia coli outer membrane phospholipase A: role of two serines in enzymatic activity; Brok RG et al.; In the outer membrane phospholipase A (OMPLA) of Escherichia coli, Ser144 has previously been identified by chemical modification as the active site serine residue . In a specific OMPLA-negative mutant strain, the pldA gene coding for OMPLA was shown to differ from the wild-type gene by a single point mutation, resulting in the substitution of Ser152 by phenylalanine . The role in catalysis of these two serine residues in OMPLA was investigated by site-directed mutagenesis . Ser144 and Ser152 were replaced one at the time by either alanine, valine, phenylalanine, threonine, or cysteine . Ser152 was furthermore replaced by asparagine . Replacement of Ser144 by cysteine resulted in 1% residual activity, whereas the other substitutions at this position yielded virtually inactive enzymes . Substitution of Ser 152 by threonine or asparagine resulted in 40% and 2% residual activity respectively, whereas all other substitutions at this position resulted in the loss of enzymatic activity . We propose that Ser144 is the nucleophile in catalysis, and that Ser152 is involved in hydrogen bonding either to the catalytic triad or in the oxyanion hole. Biochemistry, 1996 Jun 18, 35(24), 7715 - 30 Crystal structures of the binary and ternary complexes of 7 alpha-hydroxysteroid dehydrogenase from Escherichia coli; Tanaka N et al.; 7 alpha-Hydroxysteroid dehydrogenase (7 alpha-HSDH;1 EC 1.1.1.159) is an NAD+-dependent oxidoreductase belonging to the short-chain dehydrogenase/reductase (SDR) 1 family . It catalyzes the dehydrogenation of a hydroxyl group at position 7 of the steroid skeleton of bile acids . The crystal structure of the binary (complexed with NAD+) complex of 7 alpha-HSDH has been solved at 2.3 A resolution by the multiple isomorphous replacement method . The structure of the ternary complex {the enzyme complexed with NADH, 7-oxoglycochenodeoxycholic acid (as a reaction product), and possibly partially glycochenodeoxycholic acid (as a substrate)} has been determined by a difference Fourier method at 1.8 A resolution . The enzyme 7 alpha-HSDH is an alpha/beta doubly wound protein having a Rossmann-fold domain for NAD (H) binding . Upon substrate binding, large conformation changes occur at the substrate binding loop (between the beta F strand and alpha G helix) and the C-terminal segment (residues 250-255) . The variable amino acid sequences of the substrate-binding loop appear to be responsible for the wide variety of substrate specificities observed among the enzymes of the SDR family . The crystal structure of the ternary complex of 7 alpha-HSDH, which is the only structure available as the ternary complex among the enzymes of the SDR family, indicates that the highly conserved Tyr159 and Ser146 residues most probably directly interact with the hydroxyl group of the substrates although this observation cannot be definite due to an insufficiently characterized nature of the ternary complex . The strictly conserved Lys163 is hydrogen-bonded to both the 2'- and 3'-hydroxyl groups of the nicotinamide ribose of NAD(H) . We propose a new catalytic mechanism possibly common to all the enzymes belonging to the SDR family in which a tyrosine residue (Tyr159) acts as a catalytic base and a serine residue (Ser146) plays a subsidiary role of stabilizing substrate binding. Biochemistry, 1996 Jun 18, 35(24), 7692 - 704 Motion of spin-labeled side chains in T4 lysozyme . Correlation with protein structure and dynamics; Mchaourab HS et al.; Thirty single cysteine substitution mutants of T4 lysozyme have been prepared and spin-labeled with a sulfhydryl-specific nitroxide reagent in order to systematically investigate the relationship between nitroxide side-chain mobility and protein structure . The perturbation caused by replacement of a native residue with a nitroxide amino acid was assessed from the resulting changes in biological activity, circular dichroism, and free energy of folding . The nitroxide produced context-dependent changes in stability and activity similar to those observed for substitution with natural amino acids at the same site but had little effect on the circular dichroism spectra . At solvent-exposed sites, the structural perturbation appears to be small at the level of the backbone fold . Nitroxide side-chain mobility faithfully reflects the protein tertiary fold at all sites investigated . The primary determinants of nitroxide side-chain mobility are tertiary interactions and backbone dynamics . Tertiary interactions constrain the side-chain mobility to an extent closely correlated with the degree of interaction . At interhelical loop sites, the side chains have a high mobility, consistent with high crystallographic thermal factors . On the exposed surfaces of alpha-helices, the side-chain mobility is not restricted by interactions with nearest neighbor side chains but appears to be determined by backbone dynamics . An unexpected result is a striking difference between the mobility of residues near the C- and N-termini of helices . These results provide the foundation for another dimension of information in site-directed spin-labeling experiments that can be interpreted in terms of the protein tertiary fold, its equilibrium dynamics and time-dependent conformational changes. Biochemistry, 1996 Jun 18, 35(24), 7684 - 91 Structure determination of the N-terminal thioredoxin-like domain of protein disulfide isomerase using multidimensional heteronuclear 13C/15N NMR spectroscopy; Kemmink J et al.; As a first step in dissecting the structure of human protein disulfide isomerase (PDI), the structure of a fragment corresponding to the first 120 residues of its sequence has been determined using heteronuclear multidimensional NMR techniques . As expected from its primary structure homology, the fragment has the thioredoxin fold . Similarities and differences in their structures help to explain why thioredoxins are reductants, whereas PDI is an oxidant of protein thiol groups . The results confirm that PDI has a modular, multidomain structure, which will facilitate its structural and functional characterization. FEBS Lett, 1996 Jun 17, 388(2-3), 189 - 91 Increase in ADP-ribosyltransferase activity of rat T lymphocyte alloantigen RT6.1 by a single amino acid mutation; Maehama T et al.; A family of glycosylphosphatidylinositol-linked ADP-ribosyltransferases, of which cDNAs were cloned from various mammalian cells, possess a common Glu-rich motif (EEEVLIP) near their carboxyl termini . Although the first Glu in the common motif is replaced by Gln (Q207EEVLIP) in rat T lymphocyte alloantigens RT6.1 and RT6.2, the two RT6s appear to have both activities of NAD+ glycohydrolase and ADP-ribosyltransferase to a lesser extent . To investigate the significance of the Glu-rich motif in the two enzyme activities, we produced a mutant RT6.1 (Q207E), in which Gln207 was replaced by Glu, together with wild-type RT6s, in Escherichia coli . Kinetic analysis revealed that there were no marked differences in the Vmax and Km values of NAD+ glycohydrolases among the three recombinant proteins . The recombinant RT6.1 and RT6.2 displayed extremely low auto-ADP-ribosylation, although the latter modification was somewhat higher than the former . In contrast, much greater auto-modification was observed for the Q207E mutant . Moreover, the mutant could effectively ADP-ribosylate agmatine as a substrate . Thus, the single amino acid mutation of RT6.1 caused a marked increase in its ADP-ribosyltransferase activity, indicating that the Glu-rich motif near the carboxy terminus plays an important role in the enzyme activity. EMBO J, 1996 Jun 17, 15(12), 3144 - 52 The rpsO mRNA of Escherichia coli is polyadenylated at multiple sites resulting from endonucleolytic processing and exonucleolytic degradation; Haugel-Nielsen J et al.; The rpsO monocistronic messenger, encoding ribosomal protein S15, is destabilized upon polyadenylation occurring at the hairpin structure of the transcription terminator t1 . We report that mRNA fragments differing from the monocistronic transcript by their 3' termini are also polyadenylated in the absence of polynucleotide phosphorylase and RNase II . Some of these 3' extremities result from endonucleolytic cleavages by RNase E and RNase III and from exonucleolytic degradation . Most of these mRNA fragments are destabilized upon polyadenylation with the exception of the RNA species generated by RNase III . RNase E appears to reduce the amount of poly(A) added at the transcription terminator t1. Yeast, 1996 Jun 15, 12(7), 653 - 65 Functional characterization of the repeated UASINO element in the promoters of the INO1 and CHO2 genes of yeast; Koipally J et al.; In yeast, INO1 and CHO2 gene expression is subject to repression in response to inositol and choline supplementation . The response by both genes to inositol is controlled by a single set of regulatory factors and the highly conserved and repeated UASINO element (consensus: 5' CATGTGAAAT 3') that is found in multiple copies in both promoters . However, none of the native elements found in the INO1 and CHO2 promoters constitutes an exact match to the consensus element and the functionality of individual elements from these two promoters has not been tested . In this study, the function of individual putative UASINO elements from both promoters was tested by placing promoter fragments into a reporter construct which lacked a UAS element but contained the TATA element and start of transcription from the yeast CYC1 gene fused to the Escherichia coli lacZ gene . In addition, a set of oligonucleotides containing the consensus UASINO element with the first position systematically modified was also tested for UASINO function . These studies indicated that elements that contain a C or an A as the first base at the 5' end are functional to varying degrees . The majority of potential UASINO elements from the INO1 promoter were found to be inactive, whereas all of the elements from the CHO2 promoter tested were active . These results are discussed in light of the differential regulation of the two promoters. Structure, 1996 Jun 15, 4(6), 715 - 24 Crystal structure of transaldolase B from Escherichia coli suggests a circular permutation of the alpha/beta barrel within the class I aldolase family; Jia J et al.; BACKGROUND: Transaldolase is one of the enzymes in the non-oxidative branch of the pentose phosphate pathway . It transfers a C3 ketol fragment from a ketose donor to an aldose acceptor . Transaldolase, together with transketolase, creates a reversible link between the pentose phosphate pathway and glycolysis . The enzyme is of considerable interest as a catalyst in stereospecific organic synthesis and the aim of this work was to reveal the molecular architecture of transaldolase and provide insights into the structural basis of the enzymatic mechanism . RESULTS: The three-dimensional (3D) structure of recombinant transaldolase B from E . coli was determined at 1.87 A resolution . The enzyme subunit consists of a single eight-stranded alpha/beta-barrel domain . Two subunits form a dimer related by a twofold symmetry axis . The active-site residue Lys132 which forms a Schiff base with the substrate is located at the bottom of the active-site cleft . CONCLUSIONS: The 3D structure of transaldolase is similar to structures of other enzymes in the class I aldolase family . Comparison of these structures suggests that a circular permutation of the protein sequence might have occurred in transaldolase, which nevertheless results in a similar 3D structure . This observation provides evidence for a naturally occurring circular permutation in an alpha/beta-barrel protein . It appears that such genetic permutations occur more frequently during evolution than was previously thought. J Neurosci Res, 1996 Jun 15, 44(6), 606 - 11 Leukocyte chemotactic factor, a natural ligand to CD4, is expressed by lymphocytes and microglial cells of the MS plaque; Schluesener HJ et al.; The leukocyte chemotactic factor (LCF) is a proinflammatory cytokine and natural soluble ligand to the human CD4 molecule . LCF is produced by CD4+ and CD8+ T lymphocytes and is considered essential to the influx of CD4+ T lymphocytes and macrophages into an inflammatory lesion . In order to investigate the role of LCF in the multiple sclerosis (MS) lesion, we have used a synthetic gene to express LCF in E . coli and have produced monoclonal antibodies against LCF . Monoclonal antibodies are suited to demonstrate LCF in ELISAs . Western blots and paraffin-embedded tissue sections . In the MS lesion, immunopositive lymphocytes and microglial cells, notably, have been found . This is the first demonstration that LCF is present in MS lesions . Immunostaining of microglial cells is noteworthy, as these cells are strategically placed regulatory elements of CNS immunosurveillance and like other cells of the monocytic lineage express CD4 molecules . Thus, LCF might be a paracrine factor regulating T-lymphocyte chemoattraction and an autocrine molecule regulating microglial cell immune reactivity. Nucleic Acids Res, 1996 Jun 15, 24(12), 2422 - 8 Silencing of the Escherichia coli bgl promoter: effects of template supercoiling and cell extracts on promoter activity in vitro; Schnetz K et al.; Regulation of the Escherichia coli bgl promoter involves the catabolite gene activator protein (CAP) and silencer elements that are located upstream and downstream of the promoter and its CAP binding site . The promoter is kept in a repressed state by the silencer elements and other normally active CAP-dependent or -independent promoters are repressed as well when flanked by these elements . To assess the mechanism of promoter repression, single round in vitro transcription was carried out with plasmids bearing either the wild-type bgl promoter or one of two derivatives that escape repression in vivo by different mechanisms: C234 by improving the CAP binding site of the promoter and delta1 by a deletion within the upstream silencer sequence . Repression of the bgl promoter in vitro was shown to depend on template topology and the presence of cellular factors . With negatively supercoiled templates, all three promoters are transcribed to similar extents by purified E . coli RNA polymerase and no CAP dependence is apparent; with relaxed templates, transcription is CAP dependent, but the levels of transcription of the three promoters are comparable . Addition of crude cell extract to the simple transcription system leads to repression of all three promoter alleles in the absence of CAP . Repression of the mutant alleles but not of the wild-type promoter is completely relieved in the presence of the CAP-cAMP complex . The topology of the DNA template is also important in the differential regulation of these promoters . In the case of C234, repression by cell extract is completely relieved by CAP-cAMP on relaxed or negatively supercoiled templates, while complete derepression of delta1 by CAP-cAMP occurs on negatively supercoiled templates only . Repression by cell extract requires the presence of the histone-like protein H-NS . However, H-NS alone does not appear to be sufficient for specific silencing of the wild-type promoter, since repression of all three promoter alleles caused by purified H-NS protein is completely relieved by the CAP-cAMP complex . These data suggest that template topology, H-NS and other cellular factors are involved in the formation of a specific nucleoprotein structure in the bgl promoter-silencer region; the formation of this nucleoprotein structure keeps an otherwise active promoter in an inactive state. Nucleic Acids Res, 1996 Jun 15, 24(12), 2411 - 5 Pseudouridine synthases: four families of enzymes containing a putative uridine-binding motif also conserved in dUTPases and dCTP deaminases; Koonin EV; Using a combination of several methods for protein sequence comparison and motif analysis, it is shown that the four recently described pseudouridine syntheses with different specificities belong to four distinct families . Three of these families share two conserved motifs that are likely to be directly involved in catalysis . One of these motifs is detected also in two other families of enzymes that specifically bind uridine, namely deoxycitidine triphosphate deaminases and deoxyuridine triphosphatases . It is proposed that this motif is an essential part of the uridine-binding site . Two of the pseudouridine syntheses, one of which modifies the anticodon arm of tRNAs and the other is predicted to modify a portion of the large ribosomal subunit RNA belonging to the peptidyltransferase center, are encoded in all extensively sequenced genomes, including the 'minimal' genome of Mycoplasma genitalium . These particular RNA modifications and the respective enzymes are likely to be essential for the functioning of any cell. Nucleic Acids Res, 1996 Jun 15, 24(12), 2242 - 51 Location of the C-terminal domain of the RNA polymerase alpha subunit in different open complexes at the Escherichia coli galactose operon regulatory region; Belyaeva TA et al.; Hydroxyl radical footprinting has been used to study different open complexes between Escherichia coli RNA polymerase and the galactose operon regulatory region, which contains two overlapping promoters, P1 and P2 . Complexes at P1 were studied by exploiting a P2- mutant and complexes at P2 were studied with a P1-mutant . We have identified the precise location of alpha binding in both binary RNA polymerase-galP1 and RNA polymerase-P2 complexes from the effects of deletion of the C-terminal domain of the RNA polymerase alpha subunit: alpha binds to different sites at the upstream end of each complex . Transcription initiation at galP1 can be activated by the cyclic AMP receptor protein (CRP) . Addition of CRP to the RNA polymerase-galP1 complex displaces the C-terminal domain of alpha, which then binds to a different site upstream of CRP in the ternary CRP-RNA polymerase-galP1 complex . Thus, the C-terminal domain of alpha can occupy three different sites at the gal operon regulatory region . We have also examined the effect of disrupting the Activating Region of CRP on interactions between CRP and the C-terminal domain of alpha in ternary CRP-RNA polymerase-galP1 complexes . Footprinting experiments show that these substitutions interfere with the contact between CRP and alpha but do not affect the position of alpha binding to its site upstream of bound CRP. Nucleic Acids Res, 1996 Jun 15, 24(12), 2228 - 35 Interaction of mRNA with the Escherichia coli ribosome: accessibility of phosphorothioate-containing mRNA bound to ribosomes for iodine cleavage; Alexeeva EV et al.; The contacts of phosphate groups in mRNAs with ribosomes were studied . Two mRNAs were used: one mRNA contained in the middle two defined codons to construct the pre- and the post-translocational states, the other was a sequence around the initiation site of the natural cro-mRNA . Phosphorothioate nucleotides were randomly incorporated at a few A, G, U or C positions during in vitro transcription . Iodine can cleave the thioated positions if they are not shielded by ribosomal components . Only a few minor differences in iodine cleavage of ribosome bound and non-bound mRNA were observed: the nucleotide two positions upstream of the decoding codons (i.e . those codons involved in codon-anticodon interactions) showed a reduced accessibility for iodine and the nucleotide immediately following the decoding codons an enhanced accessibility in both elongating states . In initiating ribosomes where the mRNA contained a strong Shine-Dalgarno sequence, at least five phosphates were additionally slightly protected covering the Shine-Dalgarno sequence and nucleotides downstream including the initiator AUG in the P site (Al, G3, G-2, G-5 and A-7) . The low contact levels of the phosphates in the mRNA with the elongating ribosome strikingly contrast with the pronounced contact patterns previously described for tRNAs . The data obtained in this study, as well as results of previous studies, suggest that mRNA regions downstream and upstream of decoding codons form only weak contacts with ribosomal components and that the mRNA thus is mainly fixed by codon-anticodon interaction on the elongating ribosome. Eur J Biochem, 1996 Jun 15, 238(3), 683 - 9 The copper-topaquinone-phenylhydrazine-adduct geometry in Escherichia coli amine oxidase derivatized with phenylhydrazines substituted with 19F-NMR relaxation measurements; Steinebach V et al.; The copper quinoprotein amine oxidase from Escherichia coli was derivatized with phenylhydrazine, substituted with a F3C group at the ortho, meta, or para position . The derivatization of the topaquinone cofactor was verified by ultraviolet/visible spectroscopy . The reduction (with dithionite) of Cu(II) to Cu(I), which was required to obtain reference samples, was verified by EPR spectroscopy . 19F-NMR spectroscopy was carried out on the derivatized enzyme forms, and the spectra showed the line-broadening effect due to the paramagnetic Cu(II) . The distance between the Cu and the mean of the three F positions in the F3C groups was calculated by means of the Solomon-Bloembergen equation for the distance-dependent contribution of CU(II) to the transversal-relaxation time of the F resonance . Assuming that the F3C-phenylhydrazines in the enzyme are always aligned towards the Cu in the same way, four configurations can be envisaged that should be taken into account to determine the topology of the two cofactors . Based on these configurations, two spatial positions were found where the calculated distances triangulated, each of these positions having a symmetry-related counterpart above or below the topaquinone-phenylhydrazine plane . If it is assumed that the geometric positions of the phenylhydrazine and topaquinone moieties in the adduct remain the same in the derivatized enzymes, a number of minimum distances between the Cu and certain atoms in the topaquinone moiety of the adduct can be calculated (1.52 +/- 0.06 nm from the C2-O, 1.30 +/- 0.04 nm from the C4-O, and 1.26 +/- 0.04 nm from the C5-N) . However, one of the configurations yields very similar distances between the Cu and the C2-O and C4-O . Therefore, no conclusions can be made with regard to which OH group is closest to the Cu . By application of the same approach to the 19F-NMR data obtained for porcine-plasma marine oxidase {Williams, T J . & Falk, M.C.(1986) J . Biol . Chem . 261, 15949- 15954} we observed substantial differences between the topologies of the cofactors in the two enzymes . Possible reasons for this are discussed. J Clin Invest, 1996 Jun 15, 97(12), 2784 - 91 Platelet-activating factor acetylhydrolase deficiency . A missense mutation near the active site of an anti-inflammatory phospholipase; Stafforini DM et al.; Deficiency of plasma platelet-activating factor (PAF) acetylhydrolase is an autosomal recessive syndrome that has been associated with severe asthma in Japanese children . Acquired deficiency has been described in several human diseases usually associated with severe inflammation . PAF acetylhydrolase catalyzes the degradation of PAF and related phospholipids, which have proinflammatory, allergic, and prothrombotic properties . Thus, a deficiency in the degradation of these lipids should increase the susceptibility to inflammatory and allergic disorders . Miwa et al . reported that PAF acetylhydrolase activity is absent in 4% of the Japanese population, which suggests that it could be a common factor in such disorders, but the molecular basis of the defect is unknown . We show that inherited deficiency of PAF acetylhydrolase is the result of a point mutation in exon 9 and that this mutation completely abolishes enzymatic activity . This mutation is the cause of the lack of enzymatic activity as expression in E . coli of a construct harboring the mutation results in an inactive protein . This mutation as a heterozygous trait is present in 27% in the Japanese population . This finding will allow rapid identification of subjects predisposed to severe asthma and other PAF-mediated disorders. Biochem J, 1996 Jun 15, 316 ( Pt 3), 887 - 92 Reconstitution of the {4Fe-4S} cluster in FNR and demonstration of the aerobic-anaerobic transcription switch in vitro; Green J et al.; The FNR protein of Escherichia coli is a redox-responsive transcription regulator that activates and represses a family of genes required for anaerobic and aerobic metabolism . Reconstitution of wild-type FNR by anaerobic treatment with ferrous ions, cysteine and the NifS protein of Azotobacter vinelandii leads to the incorporation of two {4Fe-4S}2+ clusters per FNR dimer . The UV-visible spectrum of reconstituted FNR has a broad absorbance at 420 nm . The clusters are EPR silent under anaerobic conditions but are degraded to {3Fe-4S}+ by limited oxidation with air, and completely lost on prolonged air exposure . The association of FNR with the iron-sulphur clusters is confirmed by CD spectroscopy . Incorporation of the {4Fe-4S}2+ clusters increases site-specific DNA binding about 7-fold compared with apo-FNR . Anaerobic transcription activation and repression in vitro likewise depends on the presence of the iron-sulphur cluster, and its inactivation under aerobic conditions provides a demonstration in vitro of the FNR-mediated aerobic-anaerobic transcriptional switch. Biochem J, 1996 Jun 15, 316 ( Pt 3), 793 - 803 Expression of Drosophila trpl cRNA in Xenopus laevis oocytes leads to the appearance of a Ca2+ channel activated by Ca2+ and calmodulin, and by guanosine 5'{gamma-thio}triphosphate; Lan L et al.; The effects of expression of the Drosophila melanogaster Trpl protein, which is thought to encode a putative Ca2+ channel {Phillips, Bull and Kelly (1992) Neuron 8, 631-642}, on divalent cation inflow in Xenopus laevis oocytes were investigated . The addition of extracellular Ca2+ ({Ca2+}0) to oocytes injected with trpl cRNA and to mock-injected controls, both loaded with the fluorescent Ca2+ indicator fluo-3, induced a rapid initial and a slower sustained rate of increase in fluorescence, which were designated the initial and sustained rates of Ca2+ inflow respectively . Compared with mock-injected oocytes, trpl-cRNA-injected oocytes exhibited a higher resting cytoplasmic free Ca2+ concentration ({Ca2+}i), and higher initial and sustained rates of Ca2+ inflow in the basal (no agonist) states . The basal rate of Ca2+ inflow in trpl-cRNA-injected oocytes increased with (1) an increase in the time elapsed between injection of trpl cRNA and the measurement of Ca2+ inflow, (2) an increase in the amount of trpl cRNA injected and (3) an increase in {Ca2+}0 . Gd3+ inhibited the trpl cRNA-induced basal rate of Ca2+ inflow, with a concentration of approx . 5 microM Gd3+ giving half-maximal inhibition . Expression of trpl cRNA also caused an increase in the basal rate of Mn2+ inflow . The increases in resting {Ca2+}1 and in the basal rate of Ca2+ inflow induced by expression of trpl cRNA were inhibited by the calmodulin inhibitors W13, calmodazolium and peptide (281-309) of (Ca2+ and calmodulin)-dependent protein kinase II . A low concentration of exogenous calmodulin (introduced by microinjection) activated, and a higher concentration inhibited, the trpl cRNA-induced increase in basal rate of Ca2+ inflow . The action of the high concentration of exogenous calmodulin was reversed by W13 and calmodazolium . When rates of Ca2+ inflow in trpl-cRNA-injected oocytes were compared with those in mock-injected oocytes, the guanosine 5'-{beta-thio}diphosphate-stimulated rate was greater, the onset of thapsigargin-stimulated initial rate somewhat delayed and the inositol 1,4,5-trisphosphate-stimulated initial rate markedly inhibited . It is concluded that (1) the divalent cation channel activity of the Drosophila Trpl protein can be detected in Xenopus oocytes: (2) in the environment of the Xenopus oocyte the Trpl channel admits some Mn2+ as well as Ca2+, is activated by cytoplasmic free Ca2+ (through endogenous calmodulin) and by a trimeric GTP-binding regulatory protein, but does not appear to be activated by depletion of Ca2+ in the endoplasmic reticulum; and (3) expression of the Trpl protein inhibits the process by which the release of Ca2+ from intracellular stores activates endogenous store-activated Ca2+ channels. Biochem J, 1996 Jun 15, 316 ( Pt 3), 751 - 8 Phosphorylation of a cAMP-specific phosphodiesterase (HSPDE4B2B) by mitogen-activated protein kinase; Lenhard JM et al.; A cAMP-specific phosphodiesterase, HSPDE4B2B, was found to be phosphorylated when expressed in Sf9 cells or yeast . Deletion of amino acids 81-151 and 529-564 had no effect on the phosphorylation of HSPDE4B2B . Mass spectrometric analysis of purified HSPDE4B2B(1-564) . HSPDE4B2B(81-564) and HSPDE4B2B(152-528) showed that phosphorylation occurred predominantly on Ser487 and Ser489 . The stoicheiometry of phosphorylation was 1.2:1 (Ser487:Ser487, 489) . There was no evidence by MS for a non-phosphorylated form of HSPDE4B2B(81-564) or HSPDE4B2B(152-528) when expressed in Sf9 cells . There was no detectable phosphorylation of purified HSPDE4B2B(152-528) expressed in Escherichia coli . Radiolabelling experiments with 32P revealed that phosphorylation of HSPDE4B2B(152-528) expressed in Sf9 cells was abolished when Ser487 and Ser489 were mutated to alanines . Analysis of the amino acid sequence revealed that Ser487 and Ser489 of HSPDE4B2B conform to the consensus motifs for phosphorylation by mitogen-activated protein kinase (MAP kinase) and casein kinase II respectively . Kinetic experiments in vitro showed that MAP kinase-phosphorylated E.coli expressed and purified HSPDE4B2B(151-528) with a K(m) of 63 microM and a Vmax of 3.0 mumol/min per mg . In comparison, MAP kinase phosphorylated myelin basic protein with a Km of 26.0 microM and a Vmax of 5.5 mumol/min per mg under the same conditions . Using MS and mutational analysis we found that MAP kinase-phosphorylated E . coli expressed HSPDE4B2B(152-528) exclusively at Ser487 . These results suggest that phosphodiesterases could contribute to the cross-talk between the cAMP and MAP kinase signalling pathways. Biochem J, 1996 Jun 15, 316 ( Pt 3), 703 - 7 Synthesis and physiological activity of heterodimers comprising different splice forms of vascular endothelial growth factor and placenta growth factor; Birkenhager R et al.; Vascular endothelial growth factor (VEGF) and placenta growth factor (PIGF) are members of a dimeric-growth-factor family with angiogenic properties . VEGF is a highly potent and specific mitogen for endothelial cells, playing a vital role in angiogenesis in vivo . The role of PIGF is less clear . We expressed the monomeric splice forms VEGF-165, VEGF-121, PIGF-1 and PIGF-2 as unfused genes in Escherichia coli using the pCYTEXP expression system . In vitro dimerization experiments revealed that both homo- and hetero-dimers can be formed from these monomeric proteins . The dimers were tested for their ability to promote capillary growth in vivo and stimulate DNA synthesis in cultured human vascular endothelial cells . Heterodimers comprising different VEGF splice forms, or combinations of VEGF/PIGF splice forms, showed mitogenic activity . The results demonstrate that four different heterodimeric growth factors are likely to have as yet uncharacterized functions in vivo. FEMS Microbiol Lett, 1996 Jun 15, 140(1), 71 - 6 Analysis of the shut-off of ribosomal RNA promoters in Escherichia coli upon entering the stationary phase of growth; Aviv M et al.; Most bacterial RNA consists of stable RNA which is composed of rRNA and tRNA . We have followed by primer extension analysis the level of ribosomal RNA synthesis along the growth phases of a cell culture . A sharp drop in rRNA synthesis was observed upon the transition from the exponential to the stationary phase of growth . Our results demonstrate that an effective shut-off of rRNA synthesis occurs also in the absence of ppGpp . Mutations in the host factors Fis and H-NS, which are known to regulate rrn P1 promoters, did not affect the shut-off process of ribosomal RNA promoters . We also tested the effect of RpoS, the sigma factor known to induce a number of genes in the stationary phase . It was shown that the host factors Fis, H-NS and RpoS do not play a major role in the regulation of the shut-off process of rRNA synthesis . The results presented demonstrate that the rate of rRNA synthesis provides a sensitive measure of the growth phase of the bacterial culture. Cancer Res, 1996 Jun 15, 56(12), 2795 - 800 Identification of topoisomerase I as the cytotoxic target of the protoberberine alkaloid coralyne; Gatto B et al.; Protoberberine alkaloids (coralyne and its derivatives), which exhibit antileukemic activity in animal models, have been shown to be potent inducers of topoisomerase (topo) I-DNA cleavable complexes using purified recombinant human DNA topo I . Different from the structurally similar benzophenanthridine alkaloid nitidine (a dual poison of both topos I and II), coralyne and its derivatives have marginal poisoning activity against DNA topo II . Yeast cells expressing human DNA topo I are shown to be specifically sensitive to killing by coralyne derivatives and nitidine, suggesting that cellular DNA topo I is their cytotoxic target . Two human camptothecin-resistant cell lines, CPT-K5 and A2780/CPT-2000, which are known to express highly camptothecin-resistant topo I, are only marginally resistant to coralyne derivatives and nitidine . Purification of human topo I from Escherichia coli cells overexpressing CPT-K5 recombinant topo I has demonstrated similar marginal cross-resistance to nitidine . It seems possible to develop coralyne and nitidine derivatives as new topo I-targeted therapeutics to overcome aspects of camptothecin-related resistance. Exp Cell Res, 1996 Jun 15, 225(2), 338 - 47 AP-2.2: a novel AP-2-related transcription factor induced by retinoic acid during differentiation of P19 embryonal carcinoma cells; Oulad-Abdelghani M et al.; A 2.8-kb cDNA encoding a new transcription factor (AP-2.2) has been cloned from mouse P19 embryonal carcinoma cells, in which the corresponding mRNA begins to accumulate 30 min after retinoic acid (RA) addition . The predicted protein is 449 amino acids long and exhibits approximately 65% overall identity with other AP-2-related proteins (human AP-2, mouse AP-2alpha and beta) . A 96-amino-acid-long sequence, which is almost fully conserved between all these proteins, corresponds to the previously characterized human AP-2 DNA binding domain . Expression of AP-2.2 in Escherichia coli generated a protein that formed a specific complex with the AP-2 recognition site GCCN3GGC . AP-2.2 activated transcription from a reporter gene containing an AP-2 DNA binding site and acted synergistically with RARalpha to activate transcription from the CRABPII gene promoter . Transcriptional activation required the AP-2.2 amino-terminal region that contains a domain rich in proline and glutamine residues . The pattern of AP-2.2 expression in adult tissues, which is distinct from that of AP-2alpha, is essentially restricted to male and female gonads, to most if not all the squamous epithelia, and to several exocrine glands. Arch Biochem Biophys, 1996 Jun 15, 330(2), 373 - 9 Functional group characterization of homoserine kinase from Escherichia coli; Huo X et al.; Homoserine kinase (EC 2.7.1.39), a key enzyme in the aspartate pathway of amino acid biosynthesis in Escherichia coli, catalyzes the phosphorylation of L-homoserine to form L-homoserine phosphate . The ThrB gene coding for this enzyme has been cloned, and the enzyme has been overexpressed and purified to homogeneity with a simplified purification scheme . An examination of the pH dependence of the V/K profile for L-homoserine shows that the enzyme loses activity upon protonation of a single functional group and upon de-protonation of a second functional group, with both groups appearing to be of the cationic acid type . Incubation of the enzyme with diethylpyrocarbonate leads to the complete loss of enzyme activity . Spectral and chemical characterization of the derivatized enzyme has shown that this activity loss is caused by the modification of a histidine residue . Treatment of the enzyme with pyridoxal-5'-phosphate also results in enzyme inactivation . The spectra evidence for the formation of a Schiff base, and the complete protection afforded by substrates and inhibitors, indicate that homoserine kinase also contains a lysine that is essential for catalytic activity. Arch Biochem Biophys, 1996 Jun 15, 330(2), 329 - 41 Characterization and site-directed mutagenesis of aspen lignin-specific O-methyltransferase expressed in Escherichia coli; Meng H et al.; Aspen lignin-specific caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase (EC 1.2.1.68) was expressed in an active form in Escherichia coli using pET-23 vector . Two steps were used to purify (Phenyl Sepharose and S-adenosylhomocysteine-agarose chromatographies) enzyme to homogeneity . O-Methyl-transferase has a subunit of 40 kDa and native gradient gel electrophoresis indicated the active form is a dimer . Substrate specificity was investigated using over 20 phenolic compounds, which defined the nature of the substrate binding site and required substrate characteristics such as a hydroxyl group para to the side chain . Enzyme accommodates large substrates well if the side chain contains the trans-double bond found in lignin precursors . Kinetically S-adenosyl-L-methionine must bind before phenolic substrate; however, S-adenosyl-L-homocysteine and phenolic substrate or product can form stable complexes complicating the kinetic mechanism . The role of thiol side chain(s) in the catalytic mechanism was investigated since the enzyme is inhibited by p-chloromercuribenzoate . Of nine cysteine residues in the enzyme's sequence, only cysteine residues at positions 276 and 283 are invariant among higher plant O-methyltransferases of this class . These residues were replaced by serine and alanine, singly and in combination, using site-directed mutagenesis . All combinations of cysteine replacements at positions 276 and 283 yielded enzyme virtually as active as wild-type and all were still sensitive to thiol inhibition . We concluded that thiol(s) were not important in the catalytic mechanism of this class of O-methyltransferases and sensitivity to the large thiol inhibitor was probably due to reaction of cysteine thiol(s) near the surface which sterically hindered the active site. Blood, 1996 Jun 15, 87(12), 5144 - 51 Release of interleukin-12 in experimental Escherichia coli septic shock in baboons: relation to plasma levels of interleukin-10 and interferon-gamma; Jansen PM et al.; Interleukin (IL)-12 is thought to be a key factor for the induction of interferon gamma (IFN-gamma), a cytokine essential for the lethal effects of endotoxin . We report here on the release of the nonfunctional subunit of IL-12, p40, as well as biologically active heterodimeric IL-12, p70, after administration of a lethal (n = 5) or sublethal (n = 8) dose of live Escherichia coli to baboons . Remarkably, on lethal challenge, peak levels of p40 were observed at 3 hours that were about twofold lower than those elicited after sublethal challenge (2,813 +/- 515 pg/mL v 4,972 +/- 732 pg/mL, P < .05) . This disparity was also observed, although to a lesser extent, for IL-12 p70 antigen, of which maximum levels of 91 +/- 47 pg/mL and 151 +/- 41 pg/mL were measured 6 hours after a lethal or sublethal dose of E coli, respectively . Circulating p70 antigen correlated with IL-12 biologic activity (r = 0.869; P < .001) . When comparing lethal to sublethal conditions, lower peak levels of IL-12 on lethal E coli sharply contrasted with higher levels of other proinflammatory cytokines, such as tumor necrosis factor (TNF)-alpha, IL-1beta, IL-6, and IL-8 observed in these animals . Lower IL-12 concentrations in the lethal group may have resulted in part from the enhanced production of IL-10, a known inhibitor of IL-12 synthesis in vitro, as peak levels of this cytokine 3 hours postchallenge inversely correlated with peak levels of IL-12, in particular p40 (r = -0.802; P < .01) . Contrary to what might be expected if IFN-gamma were solely induced by IL-12, lethally challenged baboons generated threefold more IFN-gamma at 6 hours than those receiving a sublethal dose (P < .05) . Moreover, higher levels of IFN-gamma were associated with lower p40/p70 ratios, suggesting that, in agreement with observations in vitro, IFN-gamma may have preferentially upregulated the release of p70 over p40 . These data show that IL-12 is released in experimental septic shock in nonhuman primates and suggest that IL-10 and IFN-gamma are involved in the regulation of this release . Furthermore, this study indicates that the systemic release of IL-12 might be essential, but is not likely sufficient, to promote lethal production of IFN-gamma in sepsis. Blood, 1996 Jun 15, 87(12), 5032 - 9 Gene transfer into human bone marrow hematopoietic cells mediated by adenovirus vectors; Watanabe T et al.; Human bone marrow mononuclear cells (BMMNCs) and enriched CD34 positive (CD34+) cells were transduced with adenovirus vectors encoding Escherichia coli beta-galactosidase gene . Tranductions were carried out by 24-hour coincubation with adenovirus vectors at different multiplicities of infections (moi) . Efficacy of gene transfer into BM cells and expression of the gene product (ie, beta-galactosidase) were studied using X-Gal histochemical staining and flow cytometric analysis . X-Gal staining demonstrated that the percentage of positive cells at mois of 5 to 500 was 3.4% to 34.5% for BMMNCs and 6.0% to 20.0% for enriched CD34+ cells . Similar results (1.5% to 35.7% for BMMNCs and 5.4% to 24.2% for enriched CD34+ cells) were obtained with flow cytometric analysis using fluorescein di-beta-D-galactopyranoside (FDG) . Multicolor flow cytometry analysis, which included FDG, demonstrated that BM progenitors (CD34+ or CD34+CD38-), T cells (CD2+), B cells (CD19+), natural killer cells (CD56+), granulocytes, and monocytes all expressed the adenovirus transgene . To ascertain the effects of adenovirus vectors on normal BM progenitors, the numbers of colony forming unit-granulocyte/macrophage (CFU-GM), burst-forming unit-erythrocyte (BFU-E), and high-proliferative potential-colony-forming cells (HPP-CFC) after 24-hour coincubation with adenovirus vectors were determined . When BMMNCs or enriched CD34+ cells were incubated with adenovirus vectors at mois of 5 and 50, no significant differences in the numbers of CFU-GM, BFU-E, and HPP-CFC were observed compared with the uninfected control cells . However, the numbers of CFU-GM were significantly (P < .01) decreased when BMMNCs or enriched CD34+ cells were incubated with adenovirus vectors at a moi of 500, compared with the uninfected control cells . The adenovirus infected cells, purified by cell sorting for FDG expression, were capable of growing in culture and gave rise to various colonies (ie, CFU-GM, BFU-E, and HPP-CFC) . These data indicate that recombinant adenovirus vectors can be used to transfer genes to human BM hematopoietic cells with expression of the exogenous gene at a high transduction efficiency. J Mol Biol, 1996 Jun 14, 259(3), 502 - 11 Structure of the IIA domain of the mannose transporter from Escherichia coli at 1.7 angstroms resolution; Nunn RS et al.; The mannose transporter from Escherichia coli is a member of the phosphoenolpyruvate-dependent phosphotransferase system . The multi-subunit complex couples translocation across the bacterial inner membrane with phosphorylation of the solute . A functional fragment (IIA(Man), residues 2 to 133) of the membrane-associated IIAB(Man) subunit of the mannose transporter was expressed as a selenomethionine protein, and the unphosphorylated molecule was crystallized and its structure solved by X-ray crystallography . The protein consists of a central five-stranded beta-sheet covered by helices on either face . The order of the secondary structure elements is (beta alpha)4, alpha beta . Four beta-strands are arranged in a parallel manner with strand order 2134 and are linked by helices forming right-handed cross-over connections . The fifth strand that forms one edge of the sheet and runs antiparallel to the others is swapped between the subunits of the dimeric structure . Helices D and E form a helical hairpin . Histidine 10, which is transiently phosphorylated during catalysis, is located at the topological switch-point of the structure, close to the subunit interface . Its imidazole ring is hydrogen bonded to the buried side-chain of Asp67 . It is likely that Asp67 acts as a general base and thus increases the nucleophilicity of the histidine . Modeling suggests that the covalently bound phosphoryl group would be stabilized by the macrodipole of helix C . Putative interactions between IIA(Man) and the histidine-containing phosphocarrier protein are discussed. J Mol Biol, 1996 Jun 14, 259(3), 458 - 66 Catalytic mechanism of the metal-dependent fuculose aldolase from Escherichia coli as derived from the structure; Dreyer MK et al.; The structure of L-fuculose-1-phosphate aldolase in a cubic crystal form has been determined with and without the inhibitor phosphoglycolohydroxamate at 2.4 and 2.7 angstrom (1 angstrom = 0.1 nm) resolution, respectively . This inhibitor mimics the enediolate transition state of the substrate moiety dihydroxyacetone phosphate . The structures showed that dihydroxyacetone phosphate ligates the zinc ion of this metal-dependent class II aldolase with its hydroxyl and keto oxygen atoms, shifting Glu73 away from the zinc coordination sphere to a non-polar environment . At this position Glu73 accepts a proton in the initial reaction step, producing the enediolate which is then stabilized by the zinc ion . The other substrate moiety L-lactaldehyde was modeled, because no binding structure is yet available. Biochem Biophys Res Commun, 1996 Jun 14, 223(2), 434 - 8 Antisense inhibition of the RAD51 enhances radiosensitivity; Taki T et al.; The mammalian RAD51 gene is a homologue of the yeast RAD51 and E . coli RecA genes, which are involved in recombination and DNA repair . We examined the role of RAD51 protein in mouse cells using RAD51 antisense phosphorothioate oligonucleotides (ODNs) . The extraluminal delivery of 50 nM or 100 nM of antisense ODNs with lipofection to mouse cells resulted 90% suppression of RAD51 protein expression . The antisense ODNs significantly inhibited the cell growth and the treated cells became more sensitive to gamma-irradiation than the control groups . These results indicate mouse RAD51 plays an essential role in cell proliferation and radioresistant activity. Biochem Biophys Res Commun, 1996 Jun 14, 223(2), 264 - 71 The catalytic properties of human hepatitis B virus polymerase; Jeong JH et al.; The DNA-dependent DNA polymerase (DDDP) and RNA-dependent DNA polymerase (RDDP) activities of hepadnavirus polymerases are both essential for viral replication . Human hepatitis B virus (HBV) polymerase has been successfully expressed in Escherichia coli as a fusion protein in frame with maltose-binding protein . The present study was undertaken to characterize these two activities and introduce an in vitro assay system . In situ activity gel assays show that the polymerase has both types of activities . One hundred thirty-four kilodaltons of active full-length product was proteolytically cleaved into approximately 73 kDa of active fragment by proteinase K preincubation . Mutation of conserved YMDD motif also confirms that the activities were due to the recombinant polymerase and that this motif is essential to polymerase activity . Two activities of the polymerase show their optima under conditions of 1 mM (DDDP) or 0.25 mM (RDDP) of MnCl2, 400 mM KCl, 37 degrees C (DDDP) or 24 degrees C (RDDP), and pH 7.0-7.7 . Substitution of Mg2+ for Mn2+ results in reduction of processivity, which may explain why Mn2+ supports nucleotide incorporation to a higher level than Mg2+ . The polymerase is resistant to aphidicolin . Actinomycin D acts selectively on DDDP activity, whereas phosphonoformic acid inhibits both activities . The in vitro HBV polymerase assay system demonstrated herein will be useful for screening potential HBV polymerase inhibitor for the development of anti-HBV drugs. J Biol Chem, 1996 Jun 14, 271(24), 14591 - 5 Activation-modulated association of 14-3-3 proteins with Cbl in T cells; Liu YC et al.; 14-3-3 proteins have recently been implicated in the regulation of intracellular signaling pathways via their interaction with several oncogene and protooncogene products . We found recently that 14-3-3 associates with several tyrosine-phosphorylated proteins and phosphatidylinositol 3-kinase (PI3-K) in T cells . We report here the identification of the 120-kDa 14-3-3tau-binding phosphoprotein present in activated T cell lysates as Cbl, a protooncogene product of unknown function which was found recently to be a major protein-tyrosine kinase (PTK) substrate, and to interact with several signaling molecules including PI3-K, in T lymphocytes . The association between 14-3-3tau and Cbl was detected both in vitro and in intact T cells and, in contrast to Raf-1, was markedly increased following T cell activation . The use of truncated 14-3-3tau fusion proteins demonstrated that the 15 C-terminal residues are required for the association between 14-3-3 and three of its target proteins, namely, Cbl, Raf-1, and PI3-K . The findings that 14-3-3tau binds both PI3-K and Cbl, together with recent reports of an association between Cbl and PI3-K, suggest that 14-3-3 dimers play a critical role in signal transduction processes by promoting and coordinating protein-protein interactions of signaling proteins. J Biol Chem, 1996 Jun 14, 271(24), 14485 - 91 The transcriptional activity of the CCAAT-binding factor CBF is mediated by two distinct activation domains, one in the CBF-B subunit and the other in the CBF-C subunit; Coustry F et al.; CBF-A, CBF-B, and CBF-C together form the heterotrimeric mammalian CCAAT-binding factor, CBF, which binds to DNA to form a CBF-DNA complex . Here we examined the transcription activation function of CBF in an in vitro reconstituted system using the three purified recombinant CBF subunits expressed in Escherichia coli . Two of the subunits, CBF-A and CBF-C, were coexpressed and purified as a CBF-A/CBF-C complex . Addition of the three wild-type recombinant CBF subunits to EL4 cell nuclear extracts depleted of CBF stimulated transcription 5-20-fold from proalpha2(1) collagen promoters and 10-fold from the Rous sarcoma virus long terminal repeat . Two CBF deletion mutants, one containing full-length CBF-A and CBF-C and a CBF-B lacking the NH2-terminal residues 1-224, and the other containing full- length CBF-A and CBF-B and a CBF-C lacking the COOH-terminal residues 114-309, also stimulated transcription from these promoters, but the level of activation was reduced to half that obtained with the full-length CBF subunits . In contrast, a CBF deletion mutant protein containing full-length CBF-A and deleted forms of both CBF-B and CBF-C showed very little transcription activation from these promoters . Hence, this study demonstrates that the heterotrimeric CBF protein consists of two transcription activation domains, one present in CBF-B and the other in CBF-C, and that the two domains act additively in the in vitro assay . The activation domains of both CBF-B and CBF-C, which are rich in glutamine and hydrophobic residues, showed amino acid sequence similarities with each other and with the glutamine-rich activation domain of transcription factor Sp1. J Biol Chem, 1996 Jun 14, 271(24), 14045 - 54 Identification of a critical ligand binding determinant of the human erythropoietin receptor . Evidence for common ligand binding motifs in the cytokine receptor family; Middleton SA et al.; The erythropoietin receptor (EPOR) is a member of a family of cytokine and growth factor receptors that share conserved features in their extracellular and cytoplasmic domains . We have used site-specific mutagenesis within the extracellular domain of the EPOR to search for amino acid residues involved in erythropoietin (EPO) binding . Mutant proteins were expressed in bacteria as soluble EPO binding proteins (EBP) and characterized for EPO binding activity in a number of different assays . Substitution of phenylalanine at position 93 (Phe93) with alanine (F93A mutation) resulted in a drastic reduction in EPO binding in the EBP . More conservative tyrosine or tryptophan substitutions at Phe93 resulted in much less dramatic effects on EPO binding . Biophysical studies indicated that the F93A mutation does not result in gross structural alterations in the EBP . Furthermore, the F93A mutation in full-length EPOR expressed in COS cells abolished detectable EPO binding . This was not a result of processing or transport defects, since mutant receptor was present on the surface of the cells . Mutations in the region immediately around Phe93 and in residues homologous to other reported ligand binding determinants of the cytokine receptor family had small to moderate effects on EPO binding . These data indicate that Phe93 is a critical EPO binding determinant of the EPOR . Furthermore, since Phe93 aligns with critical ligand binding determinants in other receptors of the cytokine receptor family, these data suggest that receptors of this family may use common structural motifs to bind their cognate ligands. J Biol Chem, 1996 Jun 14, 271(24), 14296 - 301 The beta-galactosidase (Escherichia coli) reaction is partly facilitated by interactions of His-540 with the C6 hydroxyl of galactose; Roth NJ et al.; beta-Galactosidases with substitutions for His-540 were only poorly reactive with galactosyl substrates . However, the activity with substrates that were like galactose but did not have a C6 hydroxyl group was not decreased much as a result of such substitutions . The loss of transition state stabilization for galactosyl substrates as a result of substitution was between -15.4 and -22.8 kJ/mol but only between +0.34 and -6.5 for substrates that were identical to galactose but lacked the C6 hydroxyl . These findings indicate that an important function of His-540 is to aid in the stabilization of the transition state by forming a stable interaction with the C6 hydroxyl group . This suggestion was strengthened by the results of competitive inhibition studies showing that L-arabinolactone (a transition state analog inhibitor of beta-galactosidase without a C6 hydroxymethyl group) was bound as well by the substituted enzymes as by wild type, whereas transition state analog inhibitors that contain C6 hydroxyls (L-ribose and D-galactonolactone) were bound much more poorly by the substituted enzymes than by the wild type enzyme . Substrate analog inhibitor studies showed that His-540 was also important for binding interactions with the C6 hydroxyl group of the ground (substrate) state . The activation by Mg2+ was the same for the substituted enzymes as for the wild type, and equilibrium dialysis showed that H540F-beta-galactosidase bound Mg2+ as well as did normal beta-galactosidase . The k2 and Ks values seem to have the same pH interactions as wild type enzyme, whereas the k3 interactions are affected differently by pH in the substituted enzymes than in the wild type enzyme . The rate of the "degalactosylation" reaction was affected more by substitutions for His-540 than was the rate of the "galactosylation" reaction . All three substituted beta-galactosidases were less stable to heat than was wild type, but H540N-beta-galactosidase was somewhat more stable than the other two substituted enzymes . There were some differences in activity and inhibitory properties that resulted from the different substitutions. J Biol Chem, 1996 Jun 14, 271(24), 14353 - 60 Characterization of lactogen receptor-binding site 1 of human prolactin; Kinet S et al.; Prolactin (PRL) binds to two molecules of PRL receptor (PRLR) through two regions referred to as binding sites 1 and 2 . Although binding site 1 has been generally assigned to the pocket delimited by helix 1, helix 4, and the second half of loop 1, the residues involved in receptor binding have not yet all been precisely identified . In an earlier alanine-scanning mutational study, we identified three major binding determinants in loop 1 of human PRL (hPRL) (Goffin, V., Norman, M . & Martial, J . A.(1992) Mol . Endocrinol . 6, 1381-1392) . Here we focus on the two other regions that form binding site 1, namely helices 1 and 4 . Putative binding residues, selected on the basis of a three-dimensional model of hPRL constructed in this laboratory, were mutated to alanine, and recombinant hPRL mutants produced in Escherichia coli were tested for their ability to bind to the PRLR and to stimulate Nb2 cell proliferation . We thus identified nine single mutations (three in helix 1 and six in helix 4) whose effect was to reduce both binding and mitogenic activity by more than half as compared with wild-type hPRL, indicating the functional involvement of the corresponding residues . Adding these to the three binding determinants identified in loop 1, we now propose a complete picture of PRLR-binding site 1 of hPRL . As we earlier hypothesized, the binding site 1 determinants of hPRL differ from those of human growth hormone, a hPRL homolog. J Biol Chem, 1996 Jun 14, 271(24), 14143 - 9 Membrane insertion characteristics of the various transmembrane domains of the Escherichia coli TolQ protein; Lewin TM et al.; The Escherichia coli TolQ protein is a 230-amino acid integral cytoplasmic membrane protein required for the import of group A colicins, for infection by the filamentous phage, and for maintenance of the integrity of the bacterial envelope . TolQ is a polytopic protein with three membrane-spanning regions . The first membrane-spanning region has a 19-residue periplasmic NH2-terminal tail, while the second and third membrane-spanning segments are separated by a short 17-amino acid periplasmic loop . To study the membrane assembly of TolQ, fusions of different membrane-spanning regions were examined for their ability to insert in the absence of functional SecA or the membrane potential . Fusions containing the first membrane-spanning region plus the adjacent cytoplasmic domain and a construct containing the "hairpin loop," formed by the second and third membrane-spanning regions, insert in the absence of functional SecA . The fusion containing the second and third membrane-spanning regions required the membrane potential for insertion while the first membrane-spanning region was able to insert even in the absence of a membrane potential . Taken together, these results suggest that insertion of intact TolQ is not dependent on the Sec system, but does require the membrane potential. J Biol Chem, 1996 Jun 14, 271(24), 14405 - 11 Ku80-deficient cells exhibit excess degradation of extrachromosomal DNA; Liang F et al.; Mammalian cells possess a protein complex, termed DNA-PK, which binds to DNA double strand breaks in vitro . The complex consists of the heterodimeric Ku autoantigen and a DNA-dependent protein kinase, DNA-PKcs . Cell lines that are deficient for components of this complex are sensitive to ionizing radiation and have impaired V(D)J recombination, a site-specific recombination process . We have tested these cell lines for their ability to repair double strand breaks in transfected DNA . The xrs-6 cell line, which is deficient for the 80-kDa subunit of the Ku autoantigen, exhibited reduced stability of transfected DNA . Prior to obvious reductions in DNA stability, the levels of homologous recombination and DNA end joining were unaffected . However, the recovery of end joining products with precisely joined ends was reduced, with a concomitant increase in products containing deletions . Unlike the Ku80-deficient cells, no reduction in DNA stability was detected in DNA-PKcs-deficient scid cells . Scid cells also exhibited normal levels of homologous recombination and DNA end joining . These experiments implicate the Ku autoantigen, but not DNA-PKcs, in a direct role in protecting DNA ends from degradation. J Biol Chem, 1996 Jun 14, 271(24), 13948 - 52 Biosensor measurement of the binding of insulin-like growth factors I and II and their analogues to the insulin-like growth factor-binding protein-3; Heding A et al.; Most insulin-like growth factor (IGF) molecules in the circulation are found in a 150-kDa complex containing IGF-binding protein-3 (IGFBP-3) and an acid-labile subunit, which does not itself bind IGF . Affinities (Kd values) between 0.03 and 0.5 nM have been reported for IGF-I/IGFBP-3 binding, but no kinetic data are available . In this study we measured the high affinity binding of unlabeled IGFs and IGF analogues to recombinant unglycosylated IGFBP-3, using a BIAcoretrade mark instrument (Pharmacia Biosensor AB) . IGF-I binding showed fast association and slow non-first-order dissociation kinetics, and an equilibrium Kd of 0.23 nM . IGF-II had similar kinetics with slightly higher affinity . Analogues with mutations in the first 3 amino acids of the B-region (des(1-3) IGF-I and long IGF-I) showed 25 and 50 times lower affinity than IGF-I . Replacement of residues 28-37 by Gly-Gly-Gly-Gly or deletion of residues 29-41 in the C-region had little effect on the kinetic parameters, contrasting with the markedly impaired binding of these analogues to the IGF-I receptor . Swapping of the disulfide bridges in IGF-I and the C-region mutants decreased the affinity dramatically for IGFBP-3, primarily by decreasing the association rate . Insulin had approximately 1000 times lower affinity than IGF-I. J Biol Chem, 1996 Jun 14, 271(24), 14344 - 52 Exchanging interleukin-8 and melanoma growth-stimulating activity receptor binding specificities; Lowman HB et al.; Interleukin-8 (IL-8), a CXC chemokine, is known to bring about chemotaxis and activation of neutrophils through high affinity binding to at least two distinct receptors, receptor-A and receptor-B . The IL-8 homolog melanoma growth stimulating activity (MGSA) is also active toward neutrophils . In contrast to IL-8, MGSA binds receptor-B with high affinity and binds receptor-A with approximately 400-fold lower affinity . Using the structure of IL-8 (Clore et al.(1990) Biochemistry, 29, 1689-1696; Baldwin et al . (1991) Proc . Natl . Acad . Sci . U.S.A . 88, 502-506) and the NMR-determined structure of MGSA (Fairbrother et al . (1994) J . Mol . Biol . 242, 252-270), we designed variants of both IL-8 and MGSA to investigate the basis of specificity for binding of these chemokines to the IL-8 receptors . The most outstanding structural difference between IL-8 and MGSA lies in the loop preceding the first beta-strand . When the corresponding (shorter) loop from MGSA was swapped into IL-8, both receptor-A and receptor-B binding affinities were significantly (>300-fold) reduced . However, with additional mutations that affect packing interactions, an IL-8 variant specific for receptor-B binding was produced . Conversely, when the same loop from IL-8 was swapped into MGSA, receptor-B binding was maintained with only a approximately 30-fold reduction in receptor-A affinity . Again, mutations affecting packing of the loop yielded a MGSA variant with high affinity for both receptors, like IL-8 . Finally, we show, through point mutations in a monomeric IL-8 framework, that individual side chain substitutions can affect receptor specificity. J Biol Chem, 1996 Jun 14, 271(24), 14010 - 9 Insertional tagging, cloning, and expression of the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyltransferase gene . Use as a selectable marker for stable transformation; Donald RG et al.; A nonhomologous integration vector was used to identify the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) gene by insertional mutagenesis . Parasite mutants resistant to 6-thioxanthine arose at a frequency of approximately3 x 10(-7) . Genomic DNA flanking the insertion sites was retrieved by marker rescue and used to identify molecular clones exhibiting unambiguous homology to H(X)GPRT genes from other species . Sequence analysis of vector/genome junction sites reveals that integration of the linearized vector occurred with minimal rearrangement of either vector or target sequences, although the addition of filler DNA and small duplications or deletions of genomic sequences at the transgene termini was observed . Two differentially spliced classes of cDNA clones were identified, both of which complement hpt and gpt mutations in Escherichia coli . Kinetic analysis of purified recombinant enzyme revealed no significant differences between the two isoforms . Internally deleted clones spanning the genomic locus were used to create "knock-out" parasites, which lack all detectable HXGPRT activity . Complete activity could be restored to these knock-out mutants by transient transformation with either genomic DNA or cDNA-derived minigenes encoding both enzyme isoforms . Stable HXGPRT+ transformants were isolated under selection with mycophenolic acid, demonstrating the feasibility of HXGPRT as both a positive and negative selectable marker for stable transformation of T . gondii. J Biol Chem, 1996 Jun 14, 271(24), 14572 - 83 Amino acid substitutions in the two largest subunits of Escherichia coli RNA polymerase that suppress a defective Rho termination factor affect different parts of the transcription complex; Heisler LM et al.; Among the earliest rpoBC mutations identified are three suppressors of the conditional lethal rho allele, rho201 . These three mutations are of particular interest because, unlike rpoB8, they do not increase termination at all rho-dependent and rho-independent terminators . rpoB211 and rpoB212 both change Asn-1072 to His in conserved region H of rpoB (betaN1072H), whereas rpoC214 changes Arg-352 to Cys in conserved region C of rpoC (beta'R352C) . Both substitutions significantly reduce the overall rate of transcript elongation in vitro relative to wild-type RNA polymerase; however, they probably slow elongation for different reasons . The nucleotide triphosphate concentrations required at the T7 A1 promoter for both abortive trinucleotide synthesis and for promoter escape are much greater for betaN1072H . In contrast, beta'R352C and two adjacent substitutions (beta'G351S and beta'S350F), but not betaN1072H, formed open complexes of greatly reduced stability . The sequence in this region of beta' modestly resembles a region of Escherichia coli DNA polymerase I that contacts the phosphate backbone of DNA in co-crystals . Core determinants affecting open complex formation do not reside exclusively in beta', however, since the Rifr mutation rpoB2 in beta also dramatically destabilized open complexes . We suggest that the principal defects of the two Rho-suppressing substitutions may differ, perhaps reflecting a greater role of beta region H in nucleoside triphosphate-binding and nucleotide addition and of beta' region C in contacts to the DNA strands that could be important for translocation . Although both probably suppress rho201 by slowing RNA chain elongation, these differences may lead to terminator specificity that depends on the rate-limiting step at different sites. J Biol Chem, 1996 Jun 14, 271(24), 14035 - 40 Purification and characterization of the heat shock proteins HslV and HslU that form a new ATP-dependent protease in Escherichia coli; Yoo SJ et al.; The hslVU operon in Escherichia coli encodes two heat shock proteins, HslV, a 19-kDa protein homologous to beta-type subunits of the 20 S proteasomes, and HslU, a 50-kDa protein related to the ATPase ClpX . We have recently shown that HslV and HslU can function together as a novel ATP-dependent protease, the HslVU protease . We have now purified both proteins to apparent homogeneity from extracts of E . coli carrying the hslVU operon on a multicopy plasmid . HslU by itself cleaved ATP, and pure HslV is a weak peptidase degrading certain hydrophobic peptides . HslU dramatically stimulated peptide hydrolysis by HslV when ATP is present . With a 1:4 molar ratio of HslV to HslU, approximately a 200-fold increase in peptide hydrolysis was observed . HslV stimulated the ATPase activity of HslU 2-4-fold, but had little influence on the affinity of HslU to ATP . The nonhydrolyzable ATP analog, beta,gamma-met